Nucleic acid encoding anti-IgE antibodies

ABSTRACT

The present invention describes IgE antagonists (including variant anti-IgE antibodies) and their use in diagnosis, therapy or prophylaxis of allergic and other IgE-mediated disorders, including asthma, food allergies, hypersensitivity and anaphylactic reactions.

RELATION BACK AND PRIORITY INFORMATION

This is a continuation of U.S. Ser. No. 09/925,179, filed Aug. 8, 2001,now U.S. Pat. No. 6,914,129, which is a continuation of U.S. Ser. No.08/466,163, filed Jun. 6, 1995, now U.S. Pat. No. 6,329,509 B1, which isa division of U.S. Ser. No. 08/405,617, filed Mar. 15, 1995, nowabandoned, which is a continuation of U.S. Ser. No. 08/185,899, filedJan. 26, 1994, now abandoned, which is a 35 U.S.C. § 371 ofPCT/US92/06860, filed Aug. 14, 1992, which is a continuation-in-part ofboth U.S. Ser. No. 07/879,495, filed May 7, 1992, now abandoned and U.S.Ser. No. 07/744,768, filed Aug. 14, 1991, now abandoned; all of whichare incorporated by reference and to which application priority isclaimed under 35 U.S.C. § 120.

BACKGROUND OF THE INVENTION

This invention relates to amino acid sequence variant anti-IgEantibodies and to polypeptides containing IgE sequences, especially IgEantagonists and to polypeptides capable of differential binding to FcεRIand FcεRII.

IgE is a member of the immunoglobulin family that mediates allergicresponses such as asthma, food allergies, type 1 hypersensitivity andthe familiar sinus inflammation suffered on a widespread basis. IgE issecreted by, and expressed on the surface of, B-cells. IgE synthesizedby B-cells is anchored in the B-cell membrane by a transmembrane domainlinked to the mature IgE sequence by a short membrane binding region.IgE also is bound to B-cells (and monocytes, eosinophils and platelets)through its Fc region to a low affinity IgE receptor (FcεRII, hereafter“FCEL”). Upon exposure of a mammal to an allergen, B-cells are clonallyamplified which synthesize IgE that binds the allergen. This IgE in turnis released into the circulation by the B-cells where it is bound byB-cells (through the FCEL) and by mast cells and basophils through theso-called high affinity receptor (FcεRI, hereinafter “FCEH”) found onthe surface of the mast cells and basophils. Such mast cells andbasophils are thereby sensitized for allergen. The next exposure to theallergen cross-links the FcεRI on these cells and thus activates theirrelease of histamine and other factors which are responsible forclinical hypersensitivity and anaphylaxis.

The art has reported antibodies capable of binding to FCEL-bound IgE butnot IgE located on FCEH (see for example WO 89/00138 and U.S. Pat. No.4,940,782). These antibodies are disclosed to be clinically advantageousbecause they bind to IgE found on B-cells or circulating free in thebody, but do not bind to FCEH and thus will not activate mast cells orbasophils. In addition, various amino acid sequence variants ofimmunoglobulins are known, e.g., “chimeric” and “humanized” antibodies(see, for example, U.S. Pat. No. 4,816,567; WO 91/09968; EP 452,508; andWO 91/16927). Humanized antibodies are immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity and capacity. In some instances, Fv frameworkresidues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibody may compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. These modifications are made tofurther refine and optimize antibody performance as will be more furtherdescribed infra. Also known per se are monovalent and bispecificantibodies.

It is generally understood that FCEH, like FCEL, binds to recognitionsite(s) in the IgE constant (Fc) domain. The IgE recognition site(s) forthe two receptors are poorly defined, despite considerable effort in thepast directed to the problem.

Over the past decade several studies have been undertaken to determinewhich portion of the IgE molecule is involved in binding to FcεRI andFcεRII. Essentially three approaches have been tried. First, peptidescorresponding to specific portions of IgE sequence have been used aseither competitive inhibitors of IgE-receptor binding (Burt et al., Eur.J. Immun, 17:437-440 [1987]; Helm et al., Nature, 331:180-183 [1988];Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 [1989]; Vercelli etal., Nature, 338:649-651 [1989]; Nio et al., Peptide Chemistry, 203-208[1990]) or to elicit anti-IgE antibodies which would block IgE-receptorinteraction (Burt et al., Molec. Immun. 24:379-389 [1987]; Robertson etal., Molec. Immun., 25:103-113 [1988]; Baniyash et al., Molec. Immun.25:705-711 [1988]). The most effective competitive peptide was asequence that was 1000-fold less active than IgE (Burt et al., Eur. J.Immun., 17:437-440 [1987]).

Helm et al., Proc. Natl. Acad. Sci., 86:9465-9469 (1989) found that apeptide corresponding to IgE residues 329-409 blocked in vivosensitization of human basophil granulocytes with human IgE antibodies.Further studies indicated that residues 395-409 were not essential forbinding of the 329-409 peptide to FcεRI (Helm et al., Proc. Natl. AcadSci., 86:9465-9469 [1989]). Note that the IgE sequence variantsdescribed below had the sequence of Padlan et al., Mol. Immun., 23:1063(1986), but that the immunoglobulin residue numbers used herein arethose of Kabat et al., Sequences of Proteins of Immunological Interest(National Institutes of Health, Bethesda, Md. 1987).

Vercelli et al., Nature, 338:649-651 (1989) used recombinant IgEpeptides as well as anti-Fcε monoclonal antibodies to investigate theB-cell (FcεRII) binding site of human IgE. They concluded that theFcεRII binding site is in Fcε3 near K399-V402.

Burt et al., Eur. J. Immun., 17:437-440 (1987) investigated sevenpeptides for competition against rat IgE in binding to rat mast cells.Their most active peptide, p129, was 1000-fold less active than IgE.p129 corresponds to human sequence 439-453 which includes loop EF.Another of their peptides, p130, corresponding to residues 396-419 inthe Fcε3 domain, had no activity.

Robertson et al., Molec. Immun., 25:103-113 (1988) assessed IgE bindingby sequence-directed antibodies induced by several synthetic peptides.They concluded that the sequence defined by their ε-peptide-4(corresponding to residues 446-460), was not significantly involved inreceptor binding while the sequence defined by their ε-peptide-3(corresponding to residues 387-401), was likely to be proximal to theIgE-receptor recognition site.

Nio et al., Peptide Chemistry, 203-208 (1990) evaluated numerouspeptides with respect to their ability to inhibit histamine release byhuman basophils in vitro. Only one peptide (peptide 2, Table 1),exhibited specific inhibition; this peptide encompassed residues376-388. However, a larger peptide which incorporated this sequence(peptide 3, Table 1), had no inhibitory activity.

Second, mutations in IgE have been partially explored. Schwarzbaum etal., Eur. J. Immun., 19:1015-1023 [1989] (supra) found that a pointmutant P404H (P442H by the numbering system used herein) had 2-foldreduced affinity for FcεRI on rat basophilic leukemia (RBL) cells, butthe interpretation of this finding is controversial (Weetall et al., J.Immunol., 145:3849-3854 [1990]).

Third, chimeric molecules have been constructed. Human IgE does not bindto the murine receptor (Kulczycki Jr., et al., J. Exp. Med., 139:600-616[1974]) while rodent IgE binds to the human receptor with a reducedaffinity (Conrad, et al., J. Immun., 130:327-333 [1983]); human IgG1does not bind to IgE receptors (Weetall et al., J. Immun., 145:3849-3854[1990]). Based on these observations, several groups have constructedhuman-murine chimeras or human IgE-IgG chimeras. Weetall et al., J.Immun., 145:3849-3854 (1990) made a series of human IgG1-murine IgEchimeras and concluded that the Fcε2 and Fcε3 domains are involved inbinding murine FcεRI while the Fcε4 domain is unlikely to be involved inbinding to murine FcεRI (but may possibly be involved in binding toFcεRII). However, these conclusions are uncertain since they restprimarily on lack of binding by chimeras and three of five chimeraslacked some interchain disulfide bonds.

Nissim et al., EMBO J., 10:101-107 (1991) constructed a series ofhuman-murine IgE chimeras and measured binding to RBL cells andconcluded that the portion of IgE which binds with high affinity to thespecialized Fcε receptor on RBL cells could be assigned to Fcε3.

The results reported by these authors (e.g., Helm et al. and Burt etal.) are inconsistent. Further, in the case of anti-IgE antibodies it isdifficult to eliminate the possibility of nonspecific blocking due tosteric hindrance (Schwarzbaum et al., Eur. J. Immun., 19:1015-1023[1989]). It is apparent that considerable confusion exists in the art asto the domains of IgE Fc which are involved in the binding of IgE toFCEH or in the maintenance of IgE conformation responsible for IgEbinding to FCEH.

It is an object of this invention to identify polypeptides capable ofdifferential binding to FCEL and FCEH.

It is an object herein to determine an IgE domain which is implicated inFCEH receptor binding, but which is not involved in FCEL receptorbinding, and vice-versa.

It is another object herein to identify antagonists which are capable ofinhibiting allergic responses, including antagonists that neutralize theFCEH or FCEL receptor-binding domains of Fcε, immunoglobulin analoguesthat bind FCEL but do not bind FCEH, or that bind FCEH but not FCEL andhumanized anti-huIgE antibodies that bind to FCEL-bound IgE but not toFCEH-bound IgE or which bind to IgE but do not induce histamine releaseor degranulation of mast cells.

It is another object to provide novel polypeptides for use in the assayof Fcε receptors and for use as immunogens or for selecting anti-IgEantibodies.

SUMMARY OF THE INVENTION

We have identified domains and specific residues of IgE which play animportant role in binding IgE to its FCEL and FCEH receptors, and basedon this information we have designed polypeptides which remain capableof substantially binding to only one of these two receptors while beingsubstantially incapable of binding to the other of the receptors. Thesepolypeptides are referred to as differential binding polypeptides. Inparticular, differential binding polypeptides that bind FCEL compriseIgE sequences in which one or more residues in loop EF or the β-strand Ddomain are varied, while FCEH-binding polypeptides comprise IgEsequences in which loop AB and/or β-strand B sequences are varied.Conversely, included herein are certain polypeptides comprisingfunctional IgE loop EF and β-strand D domains but loop AB and/or βstrand B domains having reduced functionality compared to wild-type,which bind differentially to FCEH, and polypeptides comprisingfunctional loop AB and β-strand B domains but β-strand D and/or loop EFdomains having reduced functionality compared to wild-type, which binddifferentially to FCEL.

The differential binding polypeptides of this invention are sufficientlyhomologous with the amino acid sequence of an IgE heavy chain that theyretain the capability to bind FCEL or FCEH, but are varied such thatthey exhibit reduced ability to bind to one of the two receptors ascompared to native IgE. In various embodiments, the polypeptides of thisinvention additionally comprise cytotoxic polypeptides, detectablelabels, conformation-restraining groups and/or amino acid sequenceswhich are heterologous to IgE, e.g., sequences from receptors orimmunoglobulins as further described below. In other embodiments, thedifferential binding polypeptides comprise IgE sequences in addition tothe above-mentioned receptor binding domains, e.g., at least onevariable domain capable of binding a predetermined antigen. In anotherembodiment, the differential binding polypeptide is an IgE variant whichis monovalent for a predetermined antigen. In a still furtherembodiment, the differential binding polypeptide comprises an inactiveIgE variable domain, i.e., one which is incapable of binding to anyantigen, or which is devoid of a variable domain or functional CDR.

The differential binding polypeptides of this invention are useful indiagnostic procedures for IgE receptors or in the therapy ofIgE-mediated disorders such as allergies. They also are useful inpreparing antibodies capable of binding regions of IgE that participatein receptor binding.

In an embodiment of this invention, variant anti-IgE antibodies areprovided for use in diagnosis or for the therapy or prophylaxis ofallergic and other IgE-mediated disorders. In particular embodiments ofthis invention anti-IgE variant antibodies are provided in which one ormore human (recipient) light chain residues 4, 13, 19, 24, 29, 30, 33,55, 57, 58, 78, 93, 94, or 104, or heavy chain residues 24, 37, 48, 49,54, 57, 60, 61, 63, 65, 67, 69, 78, 82, 97 or 100 have been modified,preferably by substitution with the residue found in the correspondingposition in the donor (generally murine) antibody. In preferredembodiments, the selected residues are light chain 13, 19, 58, 78, or104, or heavy chain residues 48, 49, 60, 61, 63, 67, 69, 82 or 82c, andmost preferably are heavy chain residues 60, 61 or light chain residue78.

In other embodiments we provide antibodies which are capable of bindingFCEL-bound IgE but which are substantially incapable of bindingFCEH-bound IgE or inducing histamine release from mast cells orbasophils, comprising a human Kabat CDR domain into which has beensubstituted a positionally analogous residue from a Kabat CDR domain ofthe murine anti-huIgE antibodies MAE11, MAE13, MAE15 or MAE17. Alsoprovided herein are bispecific antibodies and IgE-monovalent antibodies;and humanized antibodies exhibiting an affinity for IgE which rangesfrom about 0.1 to 100 times that of MAE11.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 depicts the sequence of human IgE Fcε2 and Fcε3 (SEQ ID NO: 1).This particular sequence is from Padlan et al., Molec. Immun.,23:1063-1075 (1986). Residues are numbered according to Kabat (supra).“X” residues are included to align the Padlan IgE sequence with theKabat numbering scheme. Sequences which were altered in preparingvarious IgE mutants are underlined; bold numbers below the lines denotethe mutant number. β-strand residues are overlined; loop residues aredefined by all residues intervening between two β-strands.

FIG. 2 depicts light and heavy chain sequences for MAE11 (SEQ ID NOS: 2and 3), MAE13 (SEQ ID NOS: 4 and 5) and MAE15 (SEQ ID NOS: 6 and 7).

FIG. 3 depicts heavy and light chain sequences for HuMae11V1 (SEQ IDNOS: 8 and 9).

FIGS. 4 a and 4 b depicts the percent inhibition of IgE binding to FCELand FCEH receptors, respectively, by murine monoclonal antibody Mae11 aswell as 3 humanized variants (v1, v8 and v9).

FIGS. 5 a-5 c compare the binding of the MAE11, MAE15 and MAE17antibodies to various huIgE variants. MAE1 is provided as a controlwhich binds to both B cells and mast cell-bound IgE. The mutantsscheduled in the boxes in each figure are identified in Table 11.

DETAILED DESCRIPTION OF THE INVENTION

The IgE analogue polypeptides of this invention contain an amino acidsequence which is homologous to that of a naturally occurring IgE andhave the ability to bind specifically or differentially to FCEL or FCEHbut, in varying degree, not to both. The degree of homology of suchpolypeptides to wild-type IgE is not critical since only enough IgEsequence needs to be retained to enable the IgE to bind differentiallyor specifically to one of the two receptors. In general, thepolypeptides of this invention will be IgE Fc analogues and will beabout from 80% to 99% homologous with a polypeptide sequence of anaturally occurring IgE heavy chain Fc region. Homology is determined byconventional methods in which all substitutions are considered to benonhomologous (whether conservative or nonconservative) and in which thesequences are aligned to achieve maximal homology.

It will be understood that the IgE Fc residue numbers referred to hereinare those of Kabat. In applying the residue teachings of this inventionto other IgE Fc domains it will be necessary to compare the entirecandidate sequence with the FIG. 1 sequence in order to align theresidues and correlate the residue numbers. In addition, the identity ofcertain individual residues at any given Kabat site number may vary fromIgE to IgE due to interspecies or allelic divergence. When for exampleit is stated that substitutions are introduced at residue R383 (humanIgE) it will be understood that this means introducing a substitution atthe same site in IgE even though this same site (in loop AB) may belocated at a different residue number or may be represented in theparental or starting IgE by a residue which is different than thatdescribed by Kabat. However, for the sake of clarity and simplicity theresidue numbers and identities of the Kabat human IgE heavy chainsequences will be used herein. Note that some Kabat residues weredeleted in the Padlan sequence, in which case the Kabat numbering systemis preserved by insertion of a spacer residue designated “X” (See FIG.1).

Similarly, the Kabat system is used to designate immunoglobulin residuesused in the preparation of variant, e.g. humanized, anti-IgEimmunoglobulins such as IgG, IgE, IgA or IgD. In preferred embodimentsthe recipient human immunoglobulin site is numbered in accord with Kabatsubgroups III (V_(H)) consensus and K subgroup I (V_(L)) consensussequences. In order to determine which donor residues correspond tothese Kabat consensus residues the sequences are maximally aligned,introducing gaps as necessary, using the variable domain cysteineresidues as principal guideposts. Note that CDRs vary considerably fromantibody to antibody (and by definition will not exhibit homology withthe Kabat consensus sequences). Maximal alignment of framework residues(particularly the cysteines) frequently will require the insertion of“spacer” residues in the numbering system, to be used for the F_(v)region of the donor antibody. For example, the residue “29a” referred toinfra. This represents an extra residue found in the murine donorantibody V_(H1) CDR for which a counterpart does not exist in theconsensus sequence but whose insertion is needed to obtain maximalalignment of consensus and donor sequences. In practice, then, when ahumanized antibody (ver. 1) is prepared from this donor it will containV_(H1) with residue 29a. The differential binding polypeptides of thisinvention typically contain about from 5 to 250 residues which arehomologous to an IgE heavy chain Fc region, but ordinarily will containabout from 10 to 100 such residues. Usually, the IgE Fc3 and Fc4 regionswill be present, with the Fc3 domain providing residues directlyinvolved in receptor binding with Fc4 being present to ensureconformational integrity.

Generally, the IgE is human IgE, although animal IgE such as rat,murine, equine, bovine, feline or porcine IgE is included. As notedabove, there will be variation in the residue identities and numbers forthese IgEs compared to the FIG. 1 sequence.

FCEH and FCEL are respectively defined to be the high affinity IgEreceptor (FCεRI, Ishizaka et al., Immunochemistry, 7:687-702 [1973])found on mast cells or basophils, and the low affinity receptor (FCεRII,or CD23) found on cells involved in inflammation such as monocytes,eosinophils and platelets, as well as B-cells (Capron et al., Immun.Today, 7:15-18 [1986]). FCEH and FCEL include alleles and predeterminedamino acid sequence variants thereof which bind IgE. While FCEH containsseveral polypeptide chains, the binding of candidate polypeptides to itsalpha chain is all that needs to be assayed since the alpha chain is theportion of FCEH which binds IgE.

Differential binding means that the polypeptide will bind to one of FCELor FCEH to the extent of at least about 75% of the degree with which thehomologous native IgE binds to that receptor, but will not bind to theother receptor at more than about 20% of the degree that the homologousIgE binds to the other receptor. Binding is determined by the assays ofExample 3. Included within this invention are polypeptides that arecapable of binding to one of the two receptors to a greater degree thannative IgE.

FCEL-Specific Polypeptides

These polypeptides preferentially bind to the low affinity receptor.They typically contain Fcε3 sequences in which residues within theβ-strand D domain or loop EF have been substituted or deleted, and/or anadditional residue inserted adjacent to one of such residues. For thepurposes herein, the beta strand D domain extends from N418-X431 (FIG.1, wherein X indicates a residue omitted from U266 IgE but found in theKabat sequence) and loop EF extends from G444 to T453. A preferredFCEL-specific embodiment is mutant 6 (Table 6), in which thesubstitution of 4 residues within the human IgE heavy chain sequenceK423-R428 substantially abolished FCEH binding. Other FCEL-specificembodiments comprising EF loop variants are mutants 85, 89 and thecombination of 49, 51, 52, 83, 86 and 87. These sites (the D and EFdomains) are believed to be the principal sites involved in binding IgEto FCEL. However, those skilled in the art will be able to routinelyscreen for optimal FCEL-specific polypeptides using the methods shown inthe examples once it is understood that the beta-strand D and loop EFdomains are the prinicipal mutagenesis targets.

The preferred FCEL-specific polypeptide is one in which a residue hasbeen substituted or deleted from within the β-strand D domain or loopEF, or both. For example, four residues were substituted in generatingmutation 6, and any one or more of these substitutions may beresponsible for the loss in FCEH binding while retaining FCEL binding.As for loop EF, which is involved in both FCEL and FCEH binding, it isdesirable to screen both activities in order to select the FCEL-specificIgE variants. For example, mutant 85 (in which 9 IgE residues aresubstituted by analogously positioned IgG residues) is not detectablycapable of binding to FCEH, but does bind to FCEL (see Table 11). On theother hand, conversion of site 444 from Gly to Leu abolishes binding toeither receptor, while sites 447 and 452 are involved in binding to bothreceptors since changes at these locations prevent binding to FCEL butdo not abolish FCEH binding.

Beta-Strand D Variants for FCEL Specificity

In general, D domain substitutions will be nonconservative, i.e.,substituted residues generally will differ substantially from thosefound within the homologous native IgE in terms of charge,hydrophobicity or bulk. Typically, a maximum of 4 of 14 β-strand Ddomain residues are varied (and are usually residues 423, 424, 426and/or 428), although typically any 1 to 5 of these residues aresuitable for variation. In general, no more than 4 residues need to bevaried and optimally only one will be varied.

K423 and/or K426 are substituted with any of a residue selected from thegroup of Arg, His, Cys, Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Ile,Leu, Ser, Thr, Asp, Glu, Gln and Asn, preferably Gly, Pro, Glu, Gln andAsp and most preferably Pro or Gln.

E424 and/or E425 are substituted with any of a residue selected fromAsp, Asn, Gln, His, Lys, Arg, Cys, Met, Phe, Tyr, Trp, Pro, Gly, Ala,Val, Leu, Ile, Ser and Thr, preferably Arg, Lys, Pro, Gly and His andmost preferably Arg.

R428 and/or R422 are substituted with Cys, Met, Phe, Tyr, Trp, Pro, Gly,Ala, Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn, Gln, His, and Lys,preferably Cys, Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Ser,Thr, Asp, Glu, Asn and Gln, and most preferably Tyr.

T421 is substituted with Cys, Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val,Len, Ile, Ser, Asp, Glu, Asn, Gln, His and Lys, preferably Met, Phe,Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Asp, Glu, Asn, Gln, His and Lys,and most preferably Phe, Trp, Pro, Gly, Ala, Val, Len and Ile.

S420 is substituted with Met, Phe, Tyr, Trp, Pry, Gly, Ala, Val, Leu andIle, and preferably Pro or Gly.

X429 is substituted with any other naturally occurring amino acidresidue.

It is likely that optimal differential and FCEL binding activity will beachieved by a combination of mutations. Preferably, FCEH or FCELbinding, as the case may be, will be less than 10% of native homologousIgE, and optionally will range from undetectable to 3% of nativehomologous IgE, while binding to the other receptor ranges from at leastabout 75% of native homologous IgE to 90%, and preferably 95% to greaterthan 100%, e.g. 125%. The mutations should be as conservative aspossible, i.e., involve as modest changes in hydrophobicity, charge orbulk as possible, yet still result in a polypeptide exhibiting thesedifferential binding characteristics.

Any one or more of the β-strand D domain residues also may be deleted.Deletion of residues may possess the advantage of not introducingpotentially immunogenic sites into the IgE analogue.

Examples of candidate β-strand D domain substitutional or deletionalvariants are set forth in the following Table 1. To determine thesequence of each variant, identify the residue for each variant numberunder each site. For example, the sequence of compound 19 comprises C388E389 E390, etc.

TABLE 1 HuIgE Site AA¹ 423 K 424 E 425 E 426 K 427 Q 428 R C 19 20 37 55M 18 21 38 56 F 8, 80 22 39 57, 88 Y  7 23 40 4, 75, 83-84, 89, 97 W  624 41 58, 85 P 1, 74, 78-79, 89, 25, 97 42 59 103 G 5, 76-77 26 43 60 A12, 98-99 27, 98, 100 44, 98, 101 61, 98, 102 V 13, 97 28 45 62 L 14, 8129 46 63 I 15, 82 30 47 64 S 16 31 48 65, 103 T 17 32 49 66, 104, 105 D 9 79 50 67, 86 E 9, 94 1, 3-19, 37-54, 1-72, 74, 76-78, 51 68, 8755-72, 75, 88, 89, 80-88, 93-94, 90-93, 99, 101, 99, 100-105 102, 105 N10 33 52, 79, 84  79 69 Q 11 34 3, 54, 75, 80, 1-72, 75, 70 82-83,85-89, 77, 78, 80-95, 103-104 97-103, 105 H 83, 104 35, 78, 84 53 71 K2-4, 20-72, 75, 36, 77, 79, 94 1-2, 5-36, 55-72, 104 72, 79 85-88,91-93, 74, 76, 77-90, 100-102, 105 91, 93-95, 97, 99, 100, 102, 105 R 842, 74, 76, 80, 81 89 1-3, 5-54, 74, 76-78, 83, 85-87, 103-104 80-82,90-92, 94, 99, 100-101 Δ² 90, 95, 96 91, 95, 96 91, 96 92, 96  96 93,95, 96 ¹amino acid residues substituted into the variant ²signifies adeletion

Insertion of one or more extraneous residues adjacent to a residuewithin the β-strand D domain also falls within the scope of thisinvention. Typically, only one residue will be inserted, although from 2to 4 or more residues can be inserted adjacent to any one site withinthe domain. Smaller numbers of inserted residues will be preferred inorder to avoid the introduction of immunogenic sites. This, however, ismerely a matter of choice. In general, insertions will be made at asingle site, although insertions can be made adjacent to any two or moreβ-strand D domain residues.

Insertions typically are made between the following residues: 422 and423, 423 and 424, 424 and 425, 425 and 426, 426 and 427, 427 and 428and/or 428 and 429. The inserted residue or residues generally willexhibit charge, bulk or hydrophobicity character which is distinct fromthat of the flanking residues. For example, candidate insertions can beselected from the following Table 2.

TABLE 2 Insertion β-strand D domain site¹ Q 1, 2, 3, 4, 5, 7 or 8 D 1,2, 3, 4, 5, 6 or 7 E 1, 2, 3, 4, 5, 6 or 7 F 1, 2, 3, 4, 5, 6 or 7 W 1,2, 3, 4, 5, 6 or 7 P 1 or 2 K 2 or 3 R 2 or 3 EK 2 or 7 ER 2 or 7 DK 2or 7 DR 2 or 7 G 1 or 2 A 8 Y 6 or 7 N 1, 2, 3, 4, 5, 7 or 8 H 1, 2, 3,4, 5, 7 or 8 I 1, 2, 3, 4, 5, 7 or 8 ¹422R - site 1 - 423K - site 2 -424E - site 3 - 3425E - site 4 - 426K - site 5 - 427Q - site 6 - 428R -site 7 - 429X y - site 8. Absence of a site indicates no insertion atthat site.

The FCEL-specific polypeptides need only contain so much of the IgE FcεAB-B and loop EF domain sequences as are required to substantiallyachieve FCEL binding. This is readily determinable by preparingpolypeptides comprising the AB-B and loop EF domains and incrementallyincreasing numbers of flanking or normally interposed residues, e.g.,β-strand A (N-terminal) or loop BC, β-strand C, loop CD, β-strand D,loop DE, β-strand E, β-strand F, loop EF, loop FG, β-strand G, and Fcε4(C-terminal). In general, the entire IgE sequence from Fcε3-Fcε4 isused, although fragments of FcE3 containing the AB-B domain may besatisfactory, particularly if they contain the AB-B domain, loop EF andintervening sequence, otherwise than as varied according to theteachings herein to achieve specificity for FCEL.

The FCEL-specific polypeptides are provided as linear orconformationally restrained polypeptides. Conformational restraint isaccomplished by cross-linking the polypeptide, preferably at the N- andC-termini so as to produce a cyclic structure. In preferred embodimentsthe cyclic forms have the following structure:

-   wherein (a3-a11) is a bond or the sequence -R373-F381; a12 and a18    are hydrophobic amino    -   acid residues; a13 and a14 are basic amino acid residues; and        a15, a17 and a19 are hydrophilic amino acid residues;-   R₁ is selected from    -   (a) hydroxy,    -   (b) C₁-C₈ alkoxy,    -   (c) C₃-C₁₂ alkenoxy,    -   (d) C₆-C₁₂ arlyoxy,    -   (e) acylamino-C₁-C₈-alkoxy    -   (f) pivaloyloxyethoxy,    -   (g) C₆-Cl₁₂ aryl-C₁-C₈-alkoxy where the aryl group is        unsubstituted or substituted with one or more of the groups        nitro, halo, C₁-C₄-alkoxy, and amino;    -   (h) hydroxy substituted C₂-C₈ substituted alkoxy; and    -   (i) dihydroxy substituted C₃-C₈ alkoxy;-   R₂, R₃, R₅, R₇, R₈ are the same or different and are selected from    -   (a) hydrogen,    -   (b) C₆-C₁₂ aryl where the aryl group is unsubstituted or        substituted by one or more of the groups nitro, hydroxy, halo,        C₁-C₈ alkyl, halo-C₁-C₈ alkyl, C₁-C₈ alkoxy, amino, phenyl,        acetamido, benzamido, di-C₁-C₈ alkylamino, C₆-C₁₂ aroyl, C₁-C₈        alkanoyl, and hydroxy substituted C₁-C₈ alkyl,    -   (c) C₁-C₁₂ alkyl or alkenyl; C₃-C₁₀ cycloalkyl or C₃-C₁₂        substituted with any of halo, C₁-C₈ alkoxy, C₆-C₁₂ aryloxy,        hydroxy, amino, acetamido, C₁-C₈ alkylamino, carboxy or        carboxamide;-   R₂ and R₃, R₅ and R₆, or R₇ and R₈ may optionally and independently    be joined together to form a carbocyclic or heterocyclic ring of    from four to seven atoms where the heteroatoms are selected from O,    S, or NR₁₀ where R₁₀ is selected from:    -   hydrogen, C₁-C₈-alkyl, C₂-C₈-alkenyl, C₆-C₁₂-aryl, C₃-C¹⁰        cycloalkyl, C₆-C₁₂-aryl-C₁-C₈-alkyl, C₁-C₈-alkanoyl, and C₆-C₁₂        aroyl,-   R₄ is selected from:    -   hydrogen, C₁-C₈-alkyl, C₂-C₈-alkenyl, C₆-C₁₂-aryl, C₃-C₁₀        cycloalkyl, C₆-C₁₂-aryl-C₁-C₈-alkyl, C₁-C₈-alkanoyl, and C₆-C₁₂        aroyl;-   R₂ or R₃ may be optionally joined with R₄ to form a piperidine,    pyrrolidine or thiazolidine ring;-   X is selected from:    -   an O or S atom,    -   NR₉ wherein R₉ is hydrogen, C₁-C₈-alkyl, C₃-C₈-alkenyl, C₃-C₁₀        cycloalkyl, C₆-C₁₂-aryl, C₆-C₁₂-aryl-C₁-C₈-alkyl,        C₁-C₈-alkanoyl, or C₆-C₁₂ aroyl; C₆-C₁₂ aryl, C₁-C₈ alkanoyl,        and (CH₂)k where k is an integer from 0 to 5; and        pharmaceutically acceptable salts thereof.

As used herein and unless specified otherwise: alkyl and alkenyl denotestraight or branched, saturated or unsaturated hydrocarbon chains,respectively; C₆-C₁₂ aryl groups denote unsubstituted aromatic rings orfused aromatic rings such as, for example, phenyl or naphthyl; halodenotes F, Cl, Br, or I atoms; alkoxy denotes an alkyl group bondedthrough O to the indicated site. Examples of C₁-C₈ alkyl or C₂-C₈alkenyl groups include methyl, ethyl, propyl, isopropyl, butyl, t-butyl,pentyl, isopentyl, hexyl, vinyl, allyl, butenyl and the like; examplesof C₃-C₁₀-cycloalkyl groups include cyclopropyl, cyclopentyl,cyclohexyl, and the like; heterocyclic rings include but are not limitedto pyridyl, thienyl, furyl, indolyl, benzthienyl, imidazolyl, thiazolyl,quinolinyl and isoquinolinyl. Hydrophobic amino acid residues includenaturally occurring or synthetic residues having hydrophobic sidechains, e.g. Phe, Leu, Ile, Val, Norleu, and the like. Hydrophilic aminoacid residues include naturally occurring or synthetic residues havingcharged or uncharged hydrophilic side chains, e.g. ornithine, Ser, Thr,Tyr, His, Asp, Glu, Lys and Arg. Preferably a15, a17 and a19 areunchanged and bear normal, secondary or tertiary mono or di-hydroxysubstituted alkyl side chains. Basic residues have guanidino oramino-substituted side chains for the most part.

The AB-B domain and/or loop EF-containing, FCEL-specific polypeptides ofthis invention optionally are associated with other substances or arefused to additional polypeptide sequences. The polypeptides generallycontain only IgE-homologous sequences, although they also oralternatively are labelled for diagnostic use (employing enzymes,radioisotopes, biotin or avidin, stable free radicals, andchemiluminescent or fluorescent moeities in conventional fashion). Alsothe polypeptides are fused to non-IgE polypeptides such as cytotoxic orimmunosuppressive polypeptides, to other IgE polypeptides (e.g. Fvregions), or to polypeptides capable of binding to a predeterminedligand or antigen.

Cytotoxic polypeptides include IgG Fc effector sequences and polypeptidetoxins such as diphtheria toxin or ricin A chain (U.S. Pat. Nos.4,714,749 and 4,861,579). A preferred fusion is one in which theFCEL-specific sequence (such as that of the Fcε3-Fcε4 sequence of mutant6) is fused at its N-terminus (i.e., at approximately D360) to theC-terminus of an immunoglobulin, or an immunoglobulin fragmentterminating at the C-terminus of IgG Fcγ2 or IgG Fcγ3. Alternatively theFCEL specific polypeptide is fused to an effector IgG sequence in placeof one or both of the IgG Fv domains in analogous fashion to knownimmunoadhesins.

The polypeptides herein optionally are fused to polypeptides which arecapable of binding a predetermined antigen or ligand. Generally, theseadditional polypeptides will be IgE or other immunoglobulin Fv domains,although they optionally are heterologous polypeptides such as receptorextracellular domains (produced in the known fashion of immunoadhesions,e.g. as has been accomplished with CD4). Immunoglobulin sequences fusedto the FCEL-specific polypeptides herein include Fc or variablesequences of the heavy chains of IgG1, IgG2, IgG3, IgG4, IgE, IgM, IgDor IgA. Any FCEL-specific heavy chain fusion optionally is disulfidebonded in the ordinary fashion to heavy chains having the samespecificity (thereby forming homopolymers) or to different heavy chains(thereby forming heteropolymers), including different heavy chainshaving specificity for a different antigen. Such heteropolymeric heavychains include heavy chains which are not FCEL-specific, e.g., thesecomprise native IgE sequences which bind to FCEL and FCEH in theordinary fashion, or the heavy chains optionally include at least oneheavy chain that is FCEL specific and at least one that is FCEHspecific. Heteropolymeric heavy chains also may include the heavy chainsof non-IgE immunoglobulins, e.g., IgG, IgD, IgM and the like. Inaddition, the heavy chain hetero- or homopolymers optionally aredisulfide bonded to light chains in the fashion of nativeimmunoglobulins so as to cooperatively bind to predetermined antigen inthe usual way. Unless the heteropolymeric heavy chains comprise IgMheavy chains they generally will be heterodimeric.

In some embodiments, immunoglobulins comprising an FCEL-specificpolypeptide will also comprise an immunoglobulin variable region,preferably (if at all) an IgE Fv domain. The antigenic specificity ofthe variable region may vary widely, including those which bind haptens,or which bind polypeptides or proteins from human, animal, plant,fungal, bacterial or insect sources. The specificity may be unknown orthe variable region may have the ability to bind to a predeterminedantigen. If the immunoglobulin is to have a functional variable domain(as opposed to a deleted Fv in the case of Fce3 or Fce4 fragments) it ispreferred that it have a known antigenic specificity. Antigenicspecificity may include the ability to bind antigens associated with acytotoxic or immune response particularly lymphoid cell antigens such asCD3 or CD8, integrins, B-cell surface antigens, helper or suppressorcell surface antigens, or epitopes located in the variable region ofeffector subtypes of IgG. FCEL-specific Fc domains also are usefullyemployed in combination with F_(v) domains capable of binding aparticular allergen to which a patient is allergic. These generally arehuman IgEs directed against allergens and which contain an FCEL-specificFc domain. Alternatively, the immunoglobulin specificity is directedagainst the Fc region of effector subtypes of IgG, in this case howeverit being preferable that the FCEL-specific polypeptide not suppresscomplement binding or ADCC functions of the IgG.

The polypeptides of this invention that contain antigen or ligandbinding capability contain one or more sites capable of binding to theantigen or ligand. For example, the polypeptides herein comprise one ormore IgE or other immunoglobulin Fv domain to produce monovalent orpolyvalent immunoglobulins. For the most part such polypeptides will bemonovalent for antigen or ligand, as in the case when the immunoglobulincomprises a heavy-light chain pair that has a deleted or inactivated Fvor CDR so as to not be able to bind to antigen. Alternatively, they willbe bivalent in the predominant instance, and will be monospecific orbispecific.

In another embodiment, FCEL-specific polypeptides are covalently boundto a cytotoxic agent. For example, the polypeptide ricin D toxinisolated from the Ricinus communis plant is bound to the carboxyterminus of the Fc domain, either by chemical means or, most preferably,by production of a fusion protein using standard recombinant DNAmethods. This provides a means to selectively deliver the toxin only tocells expressing FCEL on their surfaces.

The FCEL-specific polypeptides need only contain so much of the IgE Fcεsequence as is required to substantially maintain FCEL binding. This isreadily determinable by synthesizing or expressing the product anddetermining its activity. In general, the entire IgE sequence extendingfrom Fcε2-Fcε4 can be used, although fragments containing only FcE3 andFcE4 are generally satisfactory.

In general the immunoglobulin sequences and the FCEL-specific sequencewill be derived from the same species which is to be treated with theIgE analogue. Preferably, the immunoglobulin sequences are human.

The FCEL-specific polypeptides of this invention (when employed as suchwithout fusion to non-IgE sequences) exclude the linear polypeptidesequences disclosed by Nio et al. (supra), as well as other prior artpolypeptides which include the native IgE AB-B domain or loop EF (Burtet al., supra).

FCEH-Specific Polypeptides

These polypeptides are amino acid sequence variants of IgE or itsfragments in which a residue within the AB-B or loop EF domains havebeen deleted, substituted or another residue inserted so that the AB-Bor loop EF domains are no longer capable of binding to FCEL, and whichcontain sufficient beta strand D sequence and (optionally) loop EFsequence to bind to the high affinity receptor. As disclosed above, theAB-B and loop EF domains have been implicated in binding to FCEL sincemutations in these domains have a serious impact on the binding of theIgE variants to the low affinity receptor. In particular, mutations inloop EF or the C-terminal half of the AB loop and in the N-terminal halfof beta strand B produce a divergence in IgE FCEL/FCEH specificitywherein the variant continues to bind to the high affinity receptor butlargely fails to bind to the low affinity receptor. In addition, we havefound that the IgE loop EF and the heavy chain beta strand D domainsparticipate in binding to the high affinity receptor. Therefore,FCEH-specific differential binding polypeptides will comprise at leastthe FCEH-binding sequence of beta strand D and preferably also willcontain a variant AB-B or loop EF domain sequence that bindssubstantially only to FCEH.

In preferred embodiments amino acid sequence variation is introducedinto the low affinity receptor binding functionality of the AB-B or loopEF domains. Preferably, one or more of residues I382, R383, K384, S385,T387, I388, T389, C390, R446, D447, W448, I449, E150, G151, E152 or T153are varied, although modifications optionally are introduced into loopAB N-terminal to the designated loop AB residues. Only one of R383,K384, S385, T387, T-389, or R446-T453 need be mutated, although it ispreferable to vary 1, 2 or 3 residues from each domain.

When substituted at all, I382 and/or I388 generally are independentlysubstituted with Asn, Gln, Leu, Val, His, Lys, Arg, Met, Phe, Tyr, Trp,Pro, Gly, Ala, Ser, Thr, Asp or Glu, preferably Trp, Pro, Gly, Ser, Thr,Asp or Glu. Ordinarily these two residues are not modified.

R383 typically is substituted with Cys, Met, Phe, Tyr, Trp, Pro, Gly,Ala, Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn, Gln, His, or Lys,preferably Met, Phe, Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Ser, Thr,Asp, Glu, Asn or Gln and most preferably Ala, Glu, Asp or Ser.

K384 typically is substituted with Arg, His, Cys, Met, Phe, Tyr, Trp,Pro, Gly, Ala, Val, Ile, Leu, Ser, Thr, Asp, Glu, Gln and Asn,preferably Ala, Gly, Pro, Glu, Gln or Asp and most preferably Ala, Gluor Asp.

S385 is substituted with Asp, Asn, Gln, His, Lys, Arg, Cys, Met, Phe,Tyr, Trp, Pro, Gly, Ala, Val, Leu, Ile, Glu and Thr, preferably Ala,Tyr, Val, Ile, Leu, Phe, Arg, Lys and His and most preferably Ala, Val,Ile, Leu, Phe and Tyr. When substituted, P386 usually is substituted byGly, Ala, Cys, Val, Leu, Ile, Ser, Thr, Asp, Glu, Asn, Gln, His, Lys,Arg, Phe, Tyr, or Trp, and preferably Gly, Ala, Ser, Thr, Asp, Glu, Asn,Gln, His, Lys, Arg or Trp. Ordinarily, P386 is not modified.

T387 and/or T389 generally are independently substituted by Gly, Ala,Val, Leu, Ile, Ser, Asp, Pro, Glu, Asn, Gln, His, Lys, Arg, Cys, Phe,Tyr and Trp, preferably Gly, Ala, Val, Leu, Ile, Asp, Glu, Asn, Gln,His, Lys, Arg, Phe, Tyr and Trp, and most preferably Ala.

C390 ordinarily is not substituted except when employed as a componentof a cyclizing group as shown in Formula I.

The differential FCEH-binding polypeptides of this invention willcomprise the sequence of functional FCEH-binding beta strand D and loopEF domains, as defined above. In general, it is expected that thefunctional domains need not contain all of the beta strand D or loop EFdomain residues. However, any modifications of the beta strand D domainresidues will need to be conservative, if made at all, in order topreserve FCEH binding. Since loop EF is involved in both FCEL and FCEHbinding, it likely will be necessary to screen these variants in orderto determine their activity as shown in Example 5. However, a number ofloop EF mutants already have been identified that substantially abolishFCEL binding without apparently interfering with FCEH binding, e.g.mutants 50 and 52. Thus, loop EF variants may belong in either the FCELor FCEH specific category, or may equally affect binding to eachreceptor.

A particularly preferred embodiment of a FCEH-specific polypeptide isone which contains a beta strand D domain together with additionalC-terminal sequence. The sequence of this embodiment extends from aboutT421 to about T440. Generally, the N-terminus of this embodiment is S420or T421, while the C-terminus is T440, L441 or P442. In addition, one ormore residues extraneous to this sequence are fused to its N- orC-termini. These extraneous residues are particularly useful in formingcovalent or noncovalent bonds between the N- and C-termini of thispolypeptide. The N- and/or C-termini preferably are covalently bondedthrough a side chain of a residue or through the polypeptide backbone.For example, cysteine residues are fused to the N- and C-termini and,upon oxidation, a polypeptide having a terminal disulfide bond is formedwhich joins the terminal ends of the polypeptide, therebyconformationally restraining the polypeptide. Alternatively, the alphaamino group of the polypeptide (or that of an extraneous N-terminallylocated residue) is covalently bonded to the sulfur atom of anextraneous C-terminally located cysteine residue to form thioethercyclic compounds analogous to those depicted in Formula I. Other cycliccompounds are prepared in the same fashion as described elsewhereherein. Also within the scope of this embodiment are amino acid sequencevariants of native IgE sequences corresponding to the sequence of thisembodiment. Beta strand D variants are selected to enhance binding toFCEH, while the sequence outside of the beta strand D domain need onlyretain sufficient conformational structure to properly juxtapose the N-and C-termini in substantially the same position as is the case with thenative IgE sequence.

The FCEH-specific polypeptides herein optionally comprise non-IgEpolypeptides exactly as described above for the FCEL-specificpolypeptides, except that it is not prefered that the FCEH-specificpolypeptides comprise cytotoxic functionalities. In addition,conformationally restrained (typically cyclic) polypeptides comprisingthe FCEH-binding sequence of the beta strand D domain are includedwithin the scope hereof. Such polypeptides are identical to those shownin Formula I above except that the FCEH-binding beta strand D domainreplaces the (a3)-(a19) moiety. Exemplary replacement moieties includeS420-R428, T421-N430, S420-G433 and R422-R428 (note that sequences suchas T421-N430 from U266 that omit a residue from the Kabat sequence cancontain a residue at that site or may have a deletion at the samelocation, in the latter case here the Asn residue would occupy site429).

Any one or more of the AB-B domain residues also may be deleted in orderto substantially reduce or eliminate FCEL binding. Residue deletion maybe preferred for the same reason noted above with respect to the betastrand D domain.

Examples of candidate AB-B domain substitutional or deletional variantsare set forth in the following Table 3. To determine the sequence ofeach variant, identify the residue for each variant number under eachsite. For example, the sequence of compound 98 comprises A383 A384 A385,and represents the class of mutations to which mutant 7 belongs.

TABLE 3 HuIgE Site AA¹ 350 I 351 R 352 K 353 S C 55 19 37 M 56 18 38 F57, 88 8, 80 39 Y 4, 75, 83-84, 89, 7, 73 40 97 W 58, 85 6 41 P 59 1,74, 78-79 42 G 60, 73 5, 76-77 43 A 61, 98, 102 12, 98-99 44, 98, 101 V 72 62 13, 97 45 L  73 63 14, 81 46 I  75 64 15, 82 47 S 65, 103 16 1-2,5-36, 55-72, 74, 76-91, 93-95, 97, 99-100, 102, 105 T 66, 104, 105 17 49D 67, 86  9 50 E 68, 87 89, 94 51 N  79 69 10 52, 79, 84 Q 1-71, 77, 78,80-95, 70 11, 103 3, 54, 75, 80, 82-83, 97-103, 105 85-89, 103-104 H 7183, 104 4, 53 K 104 72, 79 2-4, 20-72, 75, 85-88, 48 91-93, 100-102, 105R 1-3, 5-54, 74, 76-78, 84 73 80-82, 90-92, 94, 99-101 Δ²  96 93, 95, 9690, 95, 96 92, 96 ¹Amino acid residue substituted into the analogue²Signifies a deletion.

Insertion of one or more extraneous residues adjacent to a residuewithin the AB-B domain also falls within the scope of this invention,although substitutions or deletions are preferred. Typically, only oneresidue will be inserted, although from 2 to 4 or more residues can beinserted adjacent to any one site within the AB-B domain. Smallernumbers of inserted residues will be preferred in order to avoid theintroduction of immunogenic sites. This, however, is merely a matter ofchoice. In general, insertions will be made at a single site, althoughinsertions can be made adjacent to any two or more AB-B domain residues.

Insertions typically are made between the following residues: S385 andP386, P386 and T387, T387 and I388, and I388 and T389. The insertedresidue or residues generally will exhibit charge, bulk orhydrophobicity character which is distinct from that of the flankingresidues. For example, candidate insertions can be selected from thefollowing Table 4.

TABLE 4 Insertion AB-B domain site¹ Q 1, 2, 3, 4 or 5 D 1, 2, 3, 4 or 5E 1, 2, 3, 4 or 5 F 1, 2, 3, 4 or 5 W 1, 2, 3, 4 or 5 P 1 or 2 K 2 or 3R 2 or 3 T 3 or 4 EK 2 or 4 ER 2 or 4 DK 2 or 4 DR 2 or 4 G 1 or 2 A 5 Y3 or 4 N 1, 2, 3, 4 or 5 H 1, 2, 3, 4 or 5 I 1, 2, 3, 4 or 5 ¹I382 site1 - R383 - site 2 - K384 - site 3 - S385 - site 4 - P386 - site 5 -T387. Absence of a site indicates no insertion site.One or more of the AB-B domain residues are substituted or deleted, oradditional residues inserted adjacent to such residues. In general, nomore than 4 residues or sites are varied and optimally only one will bevaried. Variations herein include combinations of insertions, deletionsor substitutions. Excluded from the scope of FCEH specific polypeptidesare the linear IgE polypeptide fragments disclosed by Nio et al. (or thenaturally occurring sequence variants of such fragments, e.g. allelesand the like), together with any other such fragments disclosed by theprior art.Loop EF Variants

Loop EF is defined above. Loop EF variants not described in the examplesmay require screening against both FCEH and FCEL assays since loop EF isinvolved in both FCEL and FCEH binding. However, this screening will beroutine and well within the ordinary skill when following the directionsand principles herein. In general, FCEH or FCEL-binding differentialpolypeptides will comprise substitutions or deletions of (or insertionsadjacent to) one or more of residues 446, 447, 448, 449, 450, 452 and453. It should be noted that sites such as 446 and 447, while shown inthe case of Ala substitution to lead to loss of FCEL binding (Example5), also serve as sites for selecting variants which bind FCEL to agreater degree than native IgE. For the most part, however, sites 446and 447 are not prefered for introducing variants in which the objectiveis FCEL binding. For this, one should focus on the region extending fromresidue 448 to 453, and preferably residues 450, 452 and 453. Ingeneral, loop EF variants are employed with variants introduced intoloop AB—beta strand B or beta strand D or both.

R446 typically is substituted by Gly, Ala, Val, Leu, Ile, Ser, His, Lys,Met, Thr, Asp, Pro, Glu, Asn, Gln, Cys, Phe, Tyr or Trp, preferably Alafor FCEH specificity.

D447 generally is substituted by Gly, Ala, Val, Leu, Ile, Met, Cys, Ser,Thr, Pro, Glu, Asn, Gln, His, Lys, Arg, Phe, Tyr or Tip, preferably Alafor FCEH specificity.

W448 also generally is not substituted, but if so then Gly, Ala, Val,Leu, Ile, Met, Cys, Ser, Thr, Pro, Glu, Asn, Asp, Gln, His, Lys, Arg,Phe or Tyr are employed.

I449 likewise generally is not substituted, but if so then Gly, Ala,Val, Leu, Met, Cys, Ser, Thr, Pro, Glu, Asn, Asp, Gln, His, Lys, Arg,Phe, Tyr or Trp are employed.

E450 typically is substituted with Gly, Ala, Val, Ile, Leu, Met, Cys,Ser, Thr, Pro, Gln, Asn, Asp, His, Lys, Arg, Phe, Tyr or Trp, preferablyAla for FCEH specificity.

G151 generally is not substituted, but if so then Ala, Val, Leu, Met,Cys, Ser, Thr, Pro, Glu, Asn, Ile, Asp, Gln, His, Lys, Arg, Phe, Tyr orTrp are employed.

E452 also generally is substituted with Ala, Val, Leu, Met, Cys, Ser,Thr, Pro, Gly, Asn, Ile, Asp, Gln, His, Lys, Arg, Phe, Tyr or Trp.

T453 typically is substituted with Ala, Val, Leu, Met, Cys, Ser, Pro,Gly, Asn, Glu, Ile, Asp, Gln, His, Lys, Arg, Phe, Tyr, or Trp.

Exemplary IgE variants are set forth in Table 5. It will be understoodthat this table may contain variants that bind to both receptors,differentially to one or the other, or to neither receptor.

TABLE 5 HuIgE Site AA¹ 446 R 447 D 450 E 452 E 453 T C 47 46 45 44 43 M34 F 33 25 Y 41 30 W 26 36, 38 36, 38 P 49 G A 13, 17 16 12, 15 12, 1412 V 31 L 40 I 48 S 29 T 43 1-3, 5-7, 9, 10, 13-17, 24-26, 28, 33, 34,37, 39, 44-48, 50, 51 D 39 1, 2, 4-15, 17-23, 5, 8, 11, 18, 23, 27, 1,29, 30, 34, 50 42 31-45, 47, 49-52 32, 33, 35, 40, 42, 52 E 9, 20 24,29, 30 1-5, 7, 9, 10, 13, 14, 3, 4, 6, 7, 9, 10, 8, 11, 18-23, 27, 16,17, 24-28, 30, 31, 13, 15-17, 24-26, 35 34, 37, 39, 43, 44, 46, 28,31-33, 37, 39, 47, 48, 51 43, 45-49, 52 N 19, 22, 40  3 50 51 Q 10, 11,23, 35, 36,  2 52 42 H 21, 30 27 36 K 18, 28, 29, 52 28 8, 11, 18-23,27, 32 35, 40, 42 R 1-8, 12, 14-16,  7  6  5  4 24-27, 31, 32, 38,44-46, 48-51 Δ² 37 38 ¹amino acid residue substituted into the variant²signifies a deletion

Variant Anti-huIgE Antibodies

Variant anti-huIgE antibodies were produced by first obtaining a groupof murine monoclonal antibodies which were capable of binding to FCELbut not to FCEH. 8 such murine monoclonal antibodies, designated MAE10,MAE11, MAE12, MAE13, MAE14, MAE15, MAE16 and MAE17, were obtained byconventional methods involving immunizing mice with human IgE or apolypeptide consisting of residues 315-547 of huIgE and screening foranti-IgE activity.

MAE11/15 and MAE13 recognize different epitopes. It appears that theMAE13 epitope is located three-dimensionally adjacent to a key componentof the FCEH binding site of IgE (but does not directly occupy that site)since a slight amount of histamine release will occur at highconcentrations of MAE 13 suggesting that some limited antibody mediatedcrosslinking of FCEH occurs with MAE 13. MAE17 was most effective insuppressing B-cell IgE synthesis despite the fact that MAE11 and MAE13exhibited greater IgE affinity. This may be attributed to its ability tomediate complement fixation (it possessed an IgG2a isotope, thuscontaining an Fc capable of eliciting effector function).

MAE11 and MAE15 are believed to recognize the same IgE epitope. Eachantibody shared certain unusual features in its amino acid sequence. Forexample, CDR1 of the light chain of each contained 3 aspartic acidresidues. CDR3 of the heavy chains of MAE11 and MAE15 contained 3histidine residues (and contained two arginine residues, respectively).

Antibodies such as the foregoing having desired IgE bindingcharacteristics may be further modified. Such modifications fall intotwo general classes. In the first class the antibodies are modified sothat they are monovalent for IgE. This means that only one “arm” of theantibody, i.e., one light-heavy chain fork of the antibody, shall becapable of binding IgE. The remaining Fv “arm” of the antibody (or armsin the case of IgM) is specific for a second (non-IgE) antigen, is notcapable of binding any antigen, or is deleted entirely. Thus, the termIgE monovalent covers polyvalent antibodies that are monovalent for IgE.The best results may be obtained with the second alternative, since thiswould preserve the structure of the antibody most faithfully and wouldlikely confer the longest circulating half-life on the antibody.IgE-monovalent antibodies specific for FCEL bound IgE optimally willcomprise sufficient Fc domains of the heavy chains to be capable ofcomplement binding and Ig effector functions.

The second antigen recognized by one embodiment of IgE monovalentantibody is one which, when indirectly cross-linked to FCEL by theantibody herein, will not produce any toxic or deleterious response,i.e. the second antigen is not FCEH, and generally is, one which is notfound in the animal to be treated (in order to avoid undesiredabsorption of the antibody onto tissues or proteins within the body).Thus, the second antigen ordinarily will not (but may be) FCEL. However,in some circumstances the second antigen will be a protein present inthe patient to be treated, e.g. where such proteins are to serve ascarriers or depot releases for the therapeutic antibodies herein.

Such IgE-monovalent antibodies are made by methods known per se. Forexample, DNA encoding the anti-IgE Fv heavy and light chains is ligatedto DNA encoding the Fc of a human recipient antibody. In addition, DNAis provided that encodes heavy and light chains for an antibody capableof binding second antigen or an unidentified antigen, or that encodesheavy and light chain having sufficient residues deleted from the CDRsthat non-IgE antigen binding no longer can occur. A conventionalrecombinant host is transformed with all four DNAs and the productsrecovered. Assuming random chain assortment, a subpopulation of antibodyproducts will contain one arm with anti-IgE heavy and light chain and atleast another arm having specificity for second antigen or no antigen.The desired subpopulation then is purified by conventional methods,e.g., immunoaffinity absorption or by molecular sieving. Theseantibodies also can be made by reduction of the starting antibodiesfollowed by oxidative chain recombination, as has heretofore beenemployed in the preparation of monovalent antibodies (see for exampleGlennie et al., Nature 295:712 [1982]).

In addition to IgE-monovalency, in other embodiments the antibodies aremodified so that they contain a maximum proportion of human sequence(commensurate with retention of required or desired activity), i.e.,they are converted to chimeras or are humanized. In both instances thefunctional effect is to place the anti-IgE binding capability of themurine or other donor antibody into a human background to make it asnon-immunogenic as possible.

General methods are known for making chimeras and for humanizingantibodies (as noted above). A minimal amount of non-human antibodysequence is substituted into the recipient human antibody. Typically,the non-human residues are substituted into the V_(H), V_(L),V_(H)-V_(L) interface or framework of the recipient human antibody.Generally, the Kabat CDR's of the humanized antibodies are about 80% andmore typically about 90% homologous with the non-human donor CDR's. TheV_(H)-V_(L) interface and framework residues of the humanized antibody,on the other hand, are about 80%, ordinarily 90% and preferably about95% homologous with the recipient human antibody. Homology is determinedby maximal alignment of identical residues. The resulting antibody is(a) less immunogenic in humans than a murine antibody and (b) capable ofbinding to FCEL-bound huIgE but substantially incapable of binding toFCEH-bound huIgE. Such antibodies typically comprise a human antibodywhich is substituted by an amino acid residue from a complementaritydetermining region (CDR), VL-VH interface or a framework region of anon-human anti-IgE antibody which is capable of binding. One or more,and preferably all, of the nonhuman CDR's L1, L2, L3, H1, H2 or H3 aresubstituted into the human antibody recipient.

The characteristics possessed by the MAE11 antibody were preferred fortherapeutic use. Since MAE11 bound to soluble IgE, bound to MIge bearingB cells, blocked IgE binding to the low and high affinity IgE receptor,inhibited in vitro IgE production and failed to bond to IgE coatedbasophils, it was chosen as the donor antibody for humanization. Therecipient antibody was Kabat human kappa (light) subgroup I and humansubgroup III heavy chain, although it will be understood that any otherhuman antibody can be suitably employed. Surprisingly, optimal resultswere not obtained by simply substituting the murine CDRs in place of theCDRs in a recipient human antibody (FIG. 3; Table 8 infra). Instead, itwas necessary to restore donor framework hydrophobic residues such asV_(H) 78, 48, 49, 63, 67, 69; 82 or 82c, or V_(L) 13, 19, 58, 78 or 104,in order to achieve a degree of inhibition of IgE binding similar tothat of the donor antibody. While these residues function to establishthe conformation of CDRs, they generally are not exposed to the exteriorof the antibody so use of the murine residues should not exert asignificant impact on immunogenicity. Other non-CDR residues exerting aneffect on binding included V_(H)60, 61, 37, 24, and V_(H)50, 52, 58 and95 (non-CDR by Chothia), and V_(L)4, V_(L)33 (non-CDR by Chothia) andV_(L)53 (non-CDR by Chothia). The human framework hydrophobic residuesgenerally are substituted with other hydrophobic residues (especiallythose from the donor antibody) such as valine, isoleucine, leucine,phenylalanine or methionine. The remaining non-CDR residues aresubstituted with any other amino acid residue, but again preferably themurine residue found at the analogous site.

In general, the character of the anti-IgE antibody is improved bysubstituting, deleting or inserting a residue at or adjacent to V_(L)sites 30, 30b, 30d, 33, 55, 57, 58, 78, 93, 94, or 104 and/or V_(H)residues 24, 37, 48, 49, 54, 57, 60, 61, 63, 65, 67, 69, 78, 82, 82c,97, 100a or 100c.

Position V_(H)-78 is most preferably substituted with phenylalanine.However, it also is substituted with leucine, valine, isoleucine,methionine, alanine or any other residue which results in an improvementin the characteristics of the antibody (see infra).

Position V_(H)-60 is most preferably substituted with asparagine,although substitution with glutamine, histidine, lysine, arginine or anyother residue which improves the characteristics of the antibody shallfall within the scope of this invention.

Position V_(H)-61 is most preferably substituted with proline, althoughglycine, alanine, valine, leucine, isoleucine or any other residue whichresults in an improvement in the characteristics of the antibody also issuitable.

CDR residues were imported from the donor MaE11. These included fourinserts in V_(L1), 30a-30d, as well as 91-94 (V_(L3)), V_(H1) 27-29,29a, 31, 33 and 34, V_(H2)53-55, and V_(H3)97-101. V_(L) 30, 30b or 30d,as well as V_(H)97, 100a or 100c, are important in conferring on the CDRability to bind IgE.

V_(H) positions 97, 100a and 100c in humae11 (humanized Mae11) are allhistidine, and 2 are arginine in MaE15. These residues are important inIgE binding. One, two or three of these are modified by substitutionwith basic residues, particularly lysine or arginine, but also withalanine, glycine, valine, isoleucine, serine, threonine, aspartic acid,glutamic acid, asparagine, glutamine, methionine, phenylalanine,tyrosine, tryptophan or proline.

V_(L) positions 30, 30b and 30d of humae11 also are important for IgEbinding. In humae11 each of these positions are occupied by the acidicresidue, aspartic acid. They are substituted in other embodiments byglutamic acid, but also may be substituted with alanine, glycine,valine, isoleucine, serine, threonine, asparagine, glutamine,methionine, phenylalanine, tyrosine, tryptophan or proline. It is withinthe scope of this invention to reverse the charges on positions V_(L)30, 30b and 30d with those on V_(H) 97, 100a and 100c, e.g. by employingaspartic acid residues in the three V_(H) sites (2 in the case ofhumanized MaE15) and histidine in the three V_(L) sites.

Residues also may be inserted adjacent to V_(H) positions 97, 100a,100c, 61 or 61, or V_(L) residues at positions 30, 30b, 30d or 78.Inserted residues generally will be of like kind, e.g. an acid residuewould be inserted adjacent to V_(L)-30, 30b or 30d, while a basicresidue is inserted adjacent to V_(H)-97, 100 or 100c. The residues atthese sites also may be deleted.

Humanized IgE-monovalent antibodies also are included within the scopeof this invention. In this instance humanization extends to the anti-IgEarm as well, if necessary, to the remaining arm(s). Non-IgE binding armsof course can originate from human antibodies and in such case will notrequire humanization.

The foregoing variations are made by introducing mutations into the DNAencoding the precursor form of the antibody and expressing the DNA inrecombinant cell culture or the like. This is accomplished byconventional methods of site directed mutagenesis. The variants then arescreened for the desired character in assays conventional per se. In thecase of anti-huIgE, desired character includes increasing the antibodyaffinity for huIgE, increasing its capacity and specificity for FCELbound IgE, increasing the concentration of antibody required tostimulate histamine release from mast cells or basophils, reducingimmunogenicity in humans, and other improvements apparent to theordinary artisan. Optimizing these characteristics frequently willrequire balancing one improvement against another and therefore is amatter of judgment and is dependent upon the performance parametersdictated by the use intended for the antibody.

It is preferable to use a human IgG1 (or other complement fixingantibody) as the recipient immunoglobulin for humanization, although huIgG2, IgG3, IgG4, IgE, IgM, IgD or IgA also can be used as recipient.Preferably the recipient is a complement fixing IgG antibody or an IgGantibody capable of participating in ADCC.

Therapeutic, Diagnostic and Preparatory Uses

The anti-IgE antibodies herein are useful in identifying IgE amino acidsequence variants in which the FCEL or FCEH-binding domains have beenmodified. Candidate FCEL or FCEH-specific polypeptides are incubatedwith these antibodies, and analogues to which these antibodies fail tobind are selected for further evaluation, e.g., determination,respectively of their FCEH and FCEL receptor binding characteristics.Any antibody, whether of murine, human, or another animal species inorigin, or a variant thereof such as the humanized immunoglobulinsdescribed above, which has the epitopic specificity of any of antibodiesMAE10-MAE17 (especially MAE11/15, MAE13 or MAE17) will be equallyacceptable. Such antibodies are easily identified by immunizing asuitable animal or using an in vitro Fv selection system, e.g. phagemid,with IgE of the appropriate animal origin and screening the animals orproducts for antibodies having the ability to compete for IgE withMAE11/15, 13, 17 or other antibodies which map to substantially the sameepitopic site(s) as those described herein. As noted, the antibodiesdesirably are monovalent for FCEL-bound IgE when employedtherapeutically. They may be bivalent and/or bispecific when used topurify IgE from plasma, serum or recombinant cell culture.

The FCEH and FCEL-specific, differential binding polypeptides are usefulfor diagnostics and therapeutics. In in vitro diagnostic assays they areemployed as specific binding reagents in assays for FCεRI or FCεRII,respectively. The polypeptides of this invention are labelled with adetectable substance such as an enzyme, fluorescent or chemiluminescentgroup, radioisotope or a specific binding moiety that binds to adetectable substance (such as an enzyme). A typical specific bindingmoiety is an immunoglobulin variable domain which is capable of bindingto the detectable substance. FCEL and FCEH specific polypeptidescomprising immunoglobulin variable domains are described in more detailabove.

Assay systems that employ the FCEL or FCEH specific polypeptides of thisinvention are analogous to the sandwich-type systems heretoforegenerally used in the immunoassay field. Here, the specific polypeptideis employed in the same fashion as labelled antibodies directed againstantigen (the FCEL or FCEH receptor) or as an absorption agentinsolubilized on a matrix for the isolation of receptor from testsample. Redox, proteolytic, esterolytic or other conventional enzymelabels are conjugated to the polypeptides of this invention for use inconventional assay systems.

The differential binding polypeptides of this invention also are usefulfor the isolation of FCEL or FCEH from cell culture in preparing FCEL orFCEH for therapeutic or research purposes. The polypeptide is covalentlybonded or noncovalently adsorbed to a matrix such as an ion exchangeresin, an immunoaffinity column (containing an antibody capable ofbinding a polypeptide fused to the FCEH or FCEL-specific polypeptide),an immobilized antigen (where the FCEH or FCEL-specific polypeptidecomprises an immunoglobulin variable region capable of binding to theantigen) or a cyanogen bromide activated polysaccharide. The immobilizedFCEH or FCEL-specific polypeptide then is contacted with the receptorpreparation under conditions such that the receptor is bound to the FCEHor FCEL-specific polypeptide. The receptor then is eluted by changingthe pH or ionic conditions and separating the polypeptide preparationfrom the receptor.

The differential binding polypeptides herein are useful in preparingantibodies specific to the FCEH or FCEL-binding domain of IgE. Forexample, antibodies capable of binding specifically to the FCEH orFCEL-binding domains of IgE are selected by first immunizing a subjectwith IgE. Monoclonal antibodies then are selected in the ordinary wayfor native IgE binding, and the monoclonal antibodies then screened toidentify those that bind to a FCEH or FCEL-specific polypeptide of thisinvention. Preferably the FCEH or FCEL-specific polypeptide will beidentical in sequence to the corresponding sequence of the IgE used asimmunogen except, of course, for the minimal mutations need to conferFCEH or FCEL differential binding specificity. For example, the IgEmonoclonal antibodies can be selected for their inability to bind tomutation 6. If they are unable to bind to mutation 6 one can concludethat they bind to the FCEH-binding site and are therefore promising foruse in diagnostic or therapeutic procedures that depend upon an antibodythat fails to bind to FCEH-bound IgE but which binds to FCEL-bound IgE.Confirmation is obtained by determining that the antibody selected infact binds to IgE bound to FCEL. Since the selected antibody is highlyspecific for the key site(s) involved in receptor binding it is thenpossible to reduce the size of the antibody; the bulk of the antibody isnot needed for steric hinderance of the IgE-receptor interaction. Thus,it becomes feasible in allergy therapy to use anti-IgE monovalentantibodies or other anti-IgE fragments such as Fab, Fab′ and the like.

Similarly, the FCEL or FCEH-specific polypeptides are useful asimmunogens for raising antibodies capable of cross-reacting with nativeIgE only at epitopic sites outside of the domains varied in creating theFCEH or FCEL-specific polypeptides. For example, mutations 6 and 7 areuseful for raising antibodies specific for IgE epitopes except for themutated AB-B or beta strand B domains as the case may be.

The FCEH and FCEL-specific polypeptides and anti-IgE antibodies(especially those with reduced immunogenicity) are useful in therapiesfor the treatment or prophylaxis of allergies, although the FCEHspecific polypeptide subgroup which bears cytotoxic functionalities isnot considered suitable for therapy since it could lead to degranulationof mast cells and basophils. Otherwise, the polypeptides typically areadministered to a patient who is known to be sensitized to an allergen,preferably prior to an acute allergic response.

The dosages and administration route will depend upon the accessoryfunctionalities accompanying the polypeptides (e.g. cytotoxic agents,immunoglobulin effector functions, etc.), the condition of the patient(including the population of B cells or mast cells and basophils), thehalf-life of the polypeptide, the affinity of the polypeptide for itsreceptor and other parameters known to the clinician. As a general guidein the case of FCEH-specific polypeptide, one will determine from bloodtests the amount of target cells circulating in the patient anddetermine the amount of polypeptide to displace or effectively competewith endogenous IgE taking into account the population of FCEH receptorsas well as the half life and affinity of the polypeptide for FCEH. Anexcess of polypeptide calculated to be necessary to substantiallydisplace native FCEH-bound IgE over a reasonable therapeutic intervalwill then be administered. Similar analysis used to determine the dosageof anti-IgE antibody or FCEL polypeptide.

Therapeutic polypeptides are administered by intravenous intrapulmonary,intraperitoneal subcutaneous or other suitable routes. Preferably thepolypeptides are administered s.c. or i.v. over a period of about from 1to 14 days as required. In the case of FCEL-specific polypeptide oranti-FCEL-bound IgE one would determine the amount needed to inhibit,suppress or kill a substantial portion of the IgE-secreting B cellpopulation. Inhibition or suppression of the B cell population includeseither or both of reductions in IgE secretion and attenuation of thetotal number of IgE secreting B cells. Candidate doses are readilydetermined by the use of in vitro cell cultures or animal models.

Therapy of allergic disorders with anti-FCEL bound IgE and FCEL or FCEHpolypeptides optionally is accomplished with other known therapies forallergies. These include administration of gamma interferon, allergendesensitization, reduction in exposure to allergen, treatment withanti-histamines and the like.

Preparation of FCEH- and FCEL-Specific Polypeptides

The FCEH- or FCEL-specific polypeptides of this invention are made inconventional fashion, i.e., modifications of amino acid sequence areaccomplished by commonly available DNA mutagenesis methods such as PCRamplification using primers bearing the mutants, or by M13 mutagenesis,followed by expression of the mutated DNA in recombinant host cells. Thepolypeptides also can be made by Merrifield or other in vitro methods ofsynthesis if they are sufficiently small (generally, under about 100residues). However, the polypeptides preferably are made by recombinantmethods. Selection of recombinant host cells, vectors, cultureconditions and other parameters are not believed to be critical. Ingeneral, hosts, vectors and methods heretofore used in the recombinantexpression of immunoglobulins (generally, IgGs) are also useful for thepreparation of the polypeptide sequences of this invention. Preferably,mammalian cells such as myelomas, CHO, Cos, 293s and the like areemployed as hosts, and the vectors are constructed for secretoryexpression of the polypeptide. Recombinant expression systems facilitatethe preparation of functional immunoglobulin variants containing FCEL-or FCEH-specific sequences since the host cells can be transformed withDNA encoding one heavy chain containing the FCEL- or FCEH-specificsequences and one light chain, each of which contains a variable domainfor binding a first antigen, and an immunoglobulin that binds antigenand FCEL or FCEH recovered.

Similarly, the same process is used with DNA encoding in additionanother heavy chain containing the FCEL- or FCEH-specific domain andanother light chain, each of which contain a variable domain for bindinga second antigen, and a bivalent immunoglobulin recovered. Properlyassembled immunoglobulin analogues are recovered by affinitychromatography on a matrix containing the two antigen(s).

The polypeptides of this invention are recovered from lysed recombinantcell culture or (when secreted) the culture supernatant. Substantialpurification is achieved by passing cell free extracts which contain thepolypeptides over an immobilized FCEL or FCEH receptor affinity matrix.Other methods heretofore used to purify IgE or other appropriateimmunoglobulins are equally acceptable here, including immunoaffinityand (when appropriate) absorption on immobilized antigen.

Polypeptides of this invention which contain short sequences preferablyare prepared using solid-phase synthesis, e.g. the method of Merrifield,J. Am. Chem. Soc., 85:2149 (1963). However, other equivalent chemicalsyntheses known in the art are acceptable. The recombinant or in vitrosynthesized polypeptides then are cross-linked to matrices (for use indiagnostic or preparatory procedures) or are placed intoconformationally restrained structures. Known cyclizing procedures suchas those described in PCT 90/01331 or Lys/Asp cyclization usingNα-Boc-amino acids on solid-phase support with Fmoc/9-fluorenylmethyl(Ofm) side-chain protection for Lys/Asp, followed by piperidinetreatment and cyclization, are useful. Methods which depend uponcross-linking or cyclization through residue side chains may requirethat an extraneous residue be inserted at the C and/or N terminus of theAB-B or beta stand D domains, as the case may be, to provide a suitablecyclizing or cross-linking site.

Glu and Lys side chains also have been crosslinked in preparing cyclicor bicyclic peptides: the peptide is synthesized by solid phasechemistry on a p-methylbenzhydrylamine resin, the peptide is cleavedfrom the resin and deprotected. The cyclic peptide is formed usingdiphenylyphosphorylazide in diluted methylformamide. For an alternativeprocedure, see Schiller et al., Peptide Protein Res. 25:171-77 (1985).See also U.S. Pat. No. 4,547,489.

Disulfide crosslinked or cyclized peptides are generated by conventionalmethods. The method of Pelton et al., J. Med Chem., 29:2370-2375 (1986)is suitable. Also useful are thiomethylene bridges (Tetrahedron Letters25:2067-2068 (1984). See also Cody et al., J. Med Chem. 28:583(1985).The C390 residue found in the C-terminal sequence of the AB-B domain isuseful in cross-linking or cyclizing this domain.

Typically, extraneous residues which are to participate in cyclizationor cross-linking are inserted at the N- and C-termini of the chosen AB-Bor beta strand D sequence as part of the synthesis of the polypeptideprecursor to be employed in the procedure. The desired cyclic orcross-linked peptides are purified by gel filtration followed byreversed-phase high pressure liquid chromatography or other conventionalprocedures. The peptides are sterilized by 0.2 μm filtration andformulated into conventional pharmacologically acceptable vehicles.

The compounds described in this invention may be the free acid or baseor converted to salts of various inorganic and organic acids and bases.Such salts are within the scope of this invention. Examples of suchsalts include ammonium, metal salts like sodium, potassium, calcium andmagnesium; salts with organic bases likedicyclohexylamine-N-methyl-D-glucamine and the like; and salts withamino acids such as arginine or lysine. Salts with inorganic and organicacids may be like prepared, for example, using hydrochloric,hydrobromic, sulfuric, phosphoric, trifluoroacetic, methanesulfonic,maleic, fumaric and the like. Non-toxic and physiologically compatiblesalts are particularly useful although other less desirable salts mayhave use in the processes of isolation and purification.

A number of methods are useful for the preparation of the saltsdescribed above and are known to those skilled in the art. For example,reaction of the free acid or free base form of a compound of Formula Iwith one or more molar equivalents of the desired acid or base in asolvent or solvent mixture in which the salt is insoluble; or in asolvent like water after which the solvent is removed by evaporation,distillation or freeze drying. Alternatively, the free acid or base formof the product may be passed over an ion exchange resin to form thedesired salt, or one salt form of the product may be converted toanother using the same general process.

Additional pharmaceutical methods may be employed to control theduration of action of the polypeptides of this invention. Controlledrelease preparations are achieved through the use of polymers whichcomplex with or absorb the subject polypeptides. Controlled delivery isachieved by formulating the polypeptides into appropriate macromoleculararticles (for example, those prepared from polyesters, polyamino acids,polyvinyl, polypyrrolidone, ethylenevinylacetate, methlycellulose,carboxymethylcellulose, or polyamine sulfate).

Alternatively, instead of entrapping the polypeptides in polymericmatrices, it is possible to entrap these materials in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization. Hydroxymethylcellulose or gelatin microcapsules andpoly-(methylmethacrylate) microcapsules, respectively, are useful, asare in colloidal drug delivery systems (for example, liposomes, albuminmicrospheres, microemulsions, nano-particles and nanocapsules). SeeRemington's Pharmaceutical Sciences (1980).

EXAMPLE 1 Preparation of Monoclonal Antibodies to IgE

Eight monoclonal antibodies with the ability to block the binding of IgEto the FCEH were used. These monoclonal antibodies, referred to asMAE10-MAE17, were made in the following manner. Purified human IgE wasprepared from supernatants of U266B1 cells (ATCC® TIB 196) usingaffinity chromatography on a previously isolated anti-IgE antibody(Genentech MAE1, although other anti-hulge antibodies are equallyuseful). For MAE12, five BALB/c female mice, age six weeks, wereimmunized in their foot pads with 10 μg of the purified IgE in Ribi'sadjuvant. Subsequent injections were done in the same manner one andthree weeks after the initial immunizations. Three days after the finalinjection, the inguinal and popliteal lymph nodes were removed andpooled, and a single cell suspension was made by passing the tissuethrough steel gauze. For MAE14, MAE15, and MAE13 the immunizations weredone in a similar manner except that for MAE13 30 μg of IgE perinjection were used and IgE 315-547 was used as a prefusion boost; forMAE14 and MAE15 five injections of 50 μg each were used; and the IgEimmunogen for MAE17 was IgE 315-547. For MAE10 and MAE11, injectionswere given subcutaneously in two doses of 100 μg and a final booster of50 μg, and spleen cells were used for the fusions. The cells were fusedat a 4:1 ratio with mouse myeloma P3X63-Ag8.653 (ATCC® CRL 1580) in highglucose (DMEM) containing 50% w/v polyethylene glycol 4000.

Fused cells were plated at a density of 2×10⁵ per well in 96 well tissueculture plates. After 24 hours HAT selective medium(hypoxanthine/aminopterin/thymidine, Sigma Chemical Company, #H0262) wasadded. Of 1440 wells plated, 365 contained growing cells after HATselection.

Fifteen days after the fusion, supernatants were tested for the presenceof antibodies specific for human IgE using an enzyme-linkedimmunosorbent assay (ELISA). The ELISA was performed as follows, withall incubations done at room temperature. Test plates (Nunc Immunoplate)were coated for 2 hours with rat anti-mouse IgG (Boehringer Mannheim, #605-500) at 1 μg/ml in 50 Mm sodium carbonate buffer, Ph 9.6, thenblocked with 0.5% bovine serum albumin in phosphate buffered saline(PBS) for 30 minutes, then washed four times with PBS containing 0.05%Tween® 20 (PBST). Test supernatants were added and incubated two hourswith shaking, then washed four times with PBST. Human IgE (purified fromU266 cells as described above) was added at 0.5 μg/ml and incubated forone hour with shaking, then washed four times in PBST. Horseradishperoxidase conjugated goat anti-human IgE (Kirkegaard & Perry Labs, #14-10-04, 0.5 mg/ml) was added at a 1:2500 dilution and incubated forone hour, then washed four times with PBST. The plates were developed byadding 100 μl/well of a solution containing 10 mg. of o-phenylenediaminedihydrochloride (Sigma Chemical Company # P8287) and 10 μl of a 30%hydrogen peroxide solution in 25 ml of phosphate citrate buffer Ph 5.0,and incubating for 15 minutes. The reaction was stopped by adding 100μl/well of 2.5 M sulfuric acid. Data was obtained by reading the platesin an automated ELISA plate reader at an absorbance of 490 nm. ForMAE12, 365 supernatants were tested and 100 were specific for human IgE.Similar frequencies of IgE specificity were obtained when screening forthe other antibodies. All of the monoclonal antibodies described hereinwere of the IgG1 isotype except for MAE17, which was IgG2b, and MAE14,which was IgG2a.

Each of the IgE specific antibodies was further tested in cell-based andplate assays to select for antibodies which bound to IgE in such a wayas to inhibit IgE binding to FCEH and which are not capable of bindingto FCEH-bound IgE. The results of these assays are set forth in Table 6and Table 6a below.

TABLE 6 SUMMARY OF MURINE Anti-Hu IgE mAb CHARACTERISTICS PBL % BindingHistamine Amount blocking Schedule/ B-cell FCEH-bound Release² FCEH³ mAbImmunogen Dose (μg) source Isotype IgE¹ (EC50) (EC50) MaE 1 PS IgE 3 ×50 Lymph IgG1 .05 μg/ml    1 μg/ml 0.3 μg Node MaE 10 U266 IgE 2 × 100,1 × 50 Spleen IgG1 No binding at >100 μg/ml 2.5 μg 10 μg/ml MaE 11 U266IgE 2 × 100, 1 × 50 Spleen IgG1 No binding at >100 μg/ml 0.6 μg 10 μg/mlMaE 12 U266 IgE 3 × 30 Lymph IgG1 No binding at >100 μg/ml 0.8 μg Node10 μg/ml MaE 13 U266 IgE 3 × 30 Lymph IgG1 No binding at >10 μg/ml 0.6μg Node 10 μg/ml MaE 14 U266 IgE 5 × 50 Lymph IgG2a No binding at >100μg/ml 2.5 μg Node 10 μg/ml MaE 15 U266 IgE 5 × 50 Lymph IgG1 No bindingat >100 μg/ml 0.6 μg Node 10 μg/ml MaE 16 rHIgE 5 × 1 Lymph IgG1 Nobinding at >100 μg/ml 0.7 μg aa 315-547 Node 10 μg/ml MaE 17 rHIge 5 × 1Lymph IgG2b No binding at >100 μg/ml >5.0 μg  aa 315-547 Node 10 μg/ml

TABLE 6a Summary of murine Anti-Hu IgE mAb (continued) % Binding %Binding of to Membrane IgE on Blocks 1 μg IgE Inhibition IgE onFcErII(CD23) binding to of in-vitro Affinity constant U266BL IM9 FcER IIIgE for IgE⁸ mAb (EC50)⁴ (EC50)⁵ (EC 50)⁶ synthesis⁷ (Kd) MaE 1 0.4μg/ml .05 μg/ml >100 μg   (−) 5.4 × 10⁻⁸   MaE 10 0.5 μg/ml No bindingat 10 μg/ml 2.5 μg (−) 7 × 10⁻⁹ MaE 11 0.15 μg/ml  No binding at 10μg/ml 0.6 μg (+) 3 × 10⁻⁸ MaE 12 >10 μg/ml  1 μg/ml 5.0 μg (−) 4 × 10⁻⁷MaE 13   1 μg/ml No binding at 10 μg/ml 0.7 μg (++) 5 × 10⁻⁸ MaE 14   6μg/ml No binding at 10 μg/ml 2.5 μg (±) 1.4 × 10⁻⁸   MaE 15   6 μg/ml Nobinding at 10 μg/ml 0.6 μg (±) 7 × 10⁻⁸ MaE 16  10 μg/ml <.05 μg/ml   5μg (+) ND MaE 17  10 μg/ml No binding at 10 μg/ml   5 μg (++) ND1. FACS Based Assays for Analysis of Murine Anti-Human IgE Monoclonals.Screen of Murine Anti-Human IgE Monoclonal Binding to IgE on CHO 3D10(FcERI Alpha+)

a. CHO 3D10 cells (FcERI alpha chain stable transfectant; Hakimi et al.,J. Biol. Chem. 265:22079) at 5×10⁵ cells per sample are incubated withU266 IgE standard (lot no. 13068-46) at 10 μg/ml in 100 μl FACS buffer(0.1% BSA 10 mN sodium azide in PBS pH 7.4) for 30 minutes at 4° C.followed by one wash with FACS buffer. The amount of IgE binding isdetermined by incubating an aliquot of IgE loaded cells with apolyclonal FITC conjugated rabbit anti-human IgG (Accurate Chem. Co.AXL-475F, lot no 16) at 50 μg/ml for 30 minutes at 4° C. followed bythree washes with FACS buffer.

b. IgE loaded cells are incubated with 100 μl of murine anti-human IgEhybridoma supernatant (murine IgG concentration ranging from 1 to 20μg/ml) for 30 min. at 4° C. followed by one wash with FACS buffer. AGenentech monoclonal anti-human IgE (MAE1) at 10 μg/ml is used as apositive control for binding. Genentech monoclonal (MAD 6P) which doesnot recognize IgE is used at 10 μg/ml as a negative control.

c. Monoclonal binding to human IgE on CHO cells is detected byincubating cells with 20 μg/ml FITC-conjugated, affinity purified F(ab)2 Goat anti-mouse IgG (Organon Teknica cat. no. 10711-0081) for 30minutes at 4° C. followed by three washes with FACS buffer. Cells areadded to 400 μl buffer contain 2 μg/ml propidium iodide (Sigma cat no.P4170) to stain dead cells.

d. Cells are analyzed on a Becton Dickinson FACSCAN flow cytometer.Forward light scatter and 90 degree side scatter gates are set toanalyze a homogeneous population of cells. Dead cells which stain withpropidium iodide are excluded from analysis. Hybridoma supernatantswhich do not bind IgE on CHO 3D10 cells were considered candidates forfurther screening.

2. Histamine Release from Peripheral Blood Basophils:

Heparinized blood was obtained from normal donors and diluted 1:4 in amodified Tyrodes buffer (25 mM tris, 150 mM NaCl, 10 mM CaCl₂,MgCl₂, 0.3mg/ml HSA, pH 7.35) then incubated with 1 nM human IgE (ND) at 4° C. for60 minutes. Cells were then added to Tyrodes buffer containing eitherthe murine monoclonal anti-IgE Abs (10 mg/ml) or a polyclonal anti-humanantiserum as the positive control, and incubated at 37 C for 30 minutes.Cells were pelleted, histamine in supernatants was acetylated andhistamine content was determined using an RIA kit (AMAC, Inc. Wesbrook,Main). Total histamine was determined from cells subjected to severalrounds of freezed thawing. Percent histamine release was calculated asnM histamine content in supernatant—nM histamine spontaneously releaseddivided by nM total histamine in the sample.

3. Blocking of Fitc Conjugated IgE Binding to FcERI Alpha Chain.

The effect of the antibodies on IgE binding was studied by preincubatingFitc labelled IgE with the various Mae antibodies at 37° C. for 30minutes in PBS containing 0.1% BSA and 10 mM Sodium Azide pH 7.4, thenincubating the complex with 5×10⁵ 3D10 cells at 4° C. for 30 minutes.The cells were then washed three times and mean channel fluorescence at475 nM was measured. A murine anti-human IgE mAb (Mae1) which does notblock IgE binding to the FcERI alpha chain was used as a control.

4. Analysis of Murine Anti-Human IgE Binding to Membrane IgE Positive BCell U266

a. U266 B1 cells (membrane IgE+) are cultured in base mediumsupplemented with 15% head inactivated fetal calf serum (Hyclone cat No.A-1111-L), penicillin, streptomycin (100 units/ml) and L-glutamine (2mM).

b. Cells (5×10⁵/aliquot) are incubated in 100 μl FACS buffer containingmurine anti-Human IgE monoclonals at 10, 5, 1, 0.5, and 0.1 μg/ml for 30minutes on ice in 96 well round bottom microtiter plates followed by twowashes with FACS buffer. The Genentech monoclonal MAE1 is used as apositive control.

c. Cells are incubated in 100 μl FACS buffer containing 50 μg/ml (1:20stock) FITC conjugated F(ab′) 2 affinity purified goat anti-mouse IgG(Organon Teknika Cat. No. 1711-0084) for 30 minutes on ice followed bythree washes with FACS buffer. Cells are added to 400 μl FACS buffercontaining propidium iodide at 2 μg/ml to stain dead cells.

5. FACS Based Binding Assays to FcERII(CD23+) B Cell IM9

a. FACS analysis of IgE binding to FcERII(CD23) (+) B cell line IM9. TheIM9 human B cell myeloma ATCC CCL 159. (Ann. N.Y. Acad. Sci.,190:221-234 [1972]) was maintained in GIF base medium with 10% heatinactivated fetal bovine serum, penicillin, streptomycin (100 units/ml)and L-glutamine (2 mM).

b. Cells (5×10⁵ aliquot) were incubated in 100 μl of FACS buffercontaining U266 IgE standard at 2 μg/ml for 30 minutes at 4° C. in 96well microtiter plates followed by 2 washes with FACS buffer. As acontrol, cells were incubated in buffer alone or buffer containing 2μg/ml human IgG1 (Behring Diagnostics Cat. no. 400112, lot no. 801024).

c. Cells were then incubated with murine anti-human IgE monoclonals at0.1 to 10 μg/ml for 30 minutes on ice. Genentech monoclonal MAE1 wasused as a positive control.

d. Cells were incubated in 100 μl FACS buffer containing FITC conjugatedF(ab′)₂ goat anti-mouse IgG at 50 μg/ml (Organon Teknika Ca #1711-0084)for 30 minutes at 4° C. followed by 3 washes with FACS buffer.

e. Cells were added to 400 μl buffer containing propidium iodide at 2μg/ml to stain dead cells.

f. Cells were analyzed on a Becton Dickinson FACSCAN flow cytometer.Forward light scatter and 90 degree side scatter gates were set toanalyze a homogeneous population of cells and dead cells which stainedwith propidium iodide were excluded from analysis. FITC positive cells(IgE binding) were analyzed relative to cells stained with FITC rabbitanti-Human IgE alone.

g. As a positive control to determine the level of CD 23 on the surfaceof IM9 cells in each experiment, an aliquot of cells was stained withBecton Dickinson murine monoclonal Leu 20 (anti-CD23) at 10 μg/ml for 30minutes at 4° C. followed by 2 washes. The cells were then incubatedwith FITC conjugated F(ab)₂ affinity purified goat anti-murine IgG at 50μg/ml.

6. Antibody Blocking of Fitc Conjugated IgE Binding to the Low AffinityIRE Receptor.

The binding of 40 nM FITC labelled IgE to the low affinity IgE receptor(CD23) expressed on the B lymphoblast cell IM-9 was analyzed by flowcytometry on a FACSCAN flow cytometer. The effect of the antibodies onFitc IgE binding was studied by preincubating Fitc IgE with the murineanti-human antibodies at 0.1 to 10 μg/ml. chimera at 37° C. for 30minutes in PBS containing 0.1% BSA and 10 mM Sodium Azide pH 7.4, thenincubating the complex with 5×10⁵ cells at 4° C. for 30 minutes. Thecells were then washed three times and mean channel fluorescence at 475nM was measured.

7. IgE In Vitro Assay Protocol

a. Peripheral blood mononuclear cells were separated from normal donors.

b. Cells were washed extensively with phosphate buffered saline toremove as many platelets as possible.

c. Mononuclear cells were counted and resuspend in media at 1×10⁶cells/ml. (Media=DMEM+pen/strep+15% horse serum+IL-2 (25 U/ml)+IL-4 (20ng/ml)).

d. Antibodies were added at appropriate concentrations on day 0, 5, and8.

e. Cultures were incubated in 24 well Falcon tissue culture plates for14 days.

f. On day 14 supernatants were removed and assayed for IgEconcentrations by an IgE specific ELISA protocol.

8. Affinity Constant (kd) of Murine mAb for Human IgE was Determined byEquilibrium Binding (Scatchard Analysis as Follows:

a. IgE (ND and PS allotypes were iodinated by the chloramine T methodand separated from free ¹²⁵I Na with a PD10 sephadex G25 column(Pharmacia cat. no. 17-0851-01) in RIA buffer:PBS, 0.5% bovine-serumalbumin (Sigma cat. no. A-7888), 0.05% Tween 20 (Sigma cat. no. P-1379),0.01% thimerosal (Sigma cat. no. T-5125), pH 7.4. Approximately 78-95%of the post column counts were precipitated with 50% trichloroaceticacid and specific activity of iodinated IgE preparations ranged from 1.6to 13 μCi/μg assuming 70% counting efficiency.

b. A fixed concentration of ¹²⁵I IgE (approximately 5×10⁴ cpm) was addedto varying concentrations of unlabelled IgE (1 to 200 nM) in a finalvolume of 0.1 ml RIA buffer in 12×75 mm polypropylene test tubes. Murineanti-human IgE mABs (20 nM final concentration) in 0.1 ml RIA bufferwere then added for a final assay volume of 0.2 ml.

c. Samples were incubated 16-18 hours at 25° C. with continuousagitation.

d. Bound and free ¹²⁵I IgE was separated by the addition of a 0.3 mlmixture of affinity purified goat anti-mouse IgG (Boehringer Mannheimcat. no. 605 208) coupled to CN Br activated Sepharose 4B (cat No.17-0430-01) and carrier protein A sepharose (Repligen cat. No. IPA 300)in RIA buffer and incubated 1 to 2 hours at 25° C. with continuousagitation. RIA buffer (1 ml) was then added, and tubes were centrifuged5 min. 400×g. Samples were counted to determine total counts.Supernatants were aspirated with a finely drawn pasteur pipet, sampleswere recounted and bound versus free counts were calculated.

e. Scatchard analysis was performed utilizing a Fortran program(scanplot) based on the Ligand program written by P. Munson at NIH.Scatplot uses a mass action equation fitting bound as a function oftotal using the Rodbard type regression analysis.

EXAMPLE 2 Preparation of Variant IgE

Based on the model of IgE Fc by Padlan & Davies (Mol. Immunol. 23:1063(1986), which is based on the crystal structure of human IgG1 Fc(Deisenhofer, Biochem. 20:2361-2370 [1981]), a series of mutants weredesigned which could be used to test the binding of human IgE to itsreceptors. These mutants are designated Emut 1-13, and are listed inTable 7 below. The Fcε3 domain is comprised of seven β-strands whichform a β-sheet structure representative of all immunoglobulin domains;there are six loops which connect these seven β-strands. We refer tothese loops by the 2 β-strands they connect, e.g. loop AB connectsβ-stands A and B. We have constructed mutants of human IgE in which wehave substituted five of the Fcε3 domain loops with their counterpartsfrom human IgG1 (Table 7, 1-5). The sixth loop contains theglycosylation site in both IgE and IgG and hence was not altered. Onemutant, (Table 7, 6), was made by exchanging human Fcε3 β-strand D withits human IgG1 Fcgamma2 counterpart. Seven additional mutants, (Table 7,7-13), consisted of the substitution of Ala residues into Fcε3 β-strandsand a loop in Fcε2.

A human IgE gene was cloned from U266, a publicly available cell line.The gene was cloned into a previously described phagemid vectorcontaining the human cytomegalovirus enhancer and promoter, a 5′ intronand sv40 polyadenylation signal (Gorman et al., DNA and Prot. Eng.Techn., 2:3-10 [1990]). Mutagenesis was performed by the Kunkel method(T. A. Kunkel, Proc. Natl. Acad. Sci. USA, 82:488-492 (1985) usingbuffers and enzymes supplied with the BioRad Muta-gene phagemid in vitromutagenesis kit, together with oligonucleotides encoding the human IgG1sequences shown in Table 7 below. Sequences of the mutant IgE DNAs werechecked only at the site of mutation using ³⁵S dideoxy sequencing.

TABLE 7 Kabat Mu- Residue No. Human IgeE Human IgG1 tant (Structure)¹Fcε3 Seq. Fcγ2 Seq. 1 377-385 (1AB) FDLFIRKS KDTLMISRT (SEQ ID NO: 10)(SEQ ID NO: 11) 2 396-401 (1BC) APSKGT SHEDPQ (SEQ ID NO: 12) (SEQ IDNO: 13) 3 407-420 (1CD) SRASGKPVNHS YVDGVQVHNAK (SEQ ID NO: 14) (SEQ IDNO: 15) 4 444-453 (1EF) GTRDWIEGET LHQDWLDGKE (SEQ ID NO: 16) (SEQ IDNO: 17) 5 465-469 (1FG) RALM APIE (SEQ ID NO: 18) (SEQ ID NO: 19) 6423-428 (βD) KEEKQR PREQQY (SEQ ID NO: 20) (SEQ ID NO: 21) 7 383-385(1AB) RKS [AAA]² 8 387, 389 (βB) T(I)T [A(I)A]² 9 403, 405 (βC) N(L)T[A(L)A]² 10 438-440 (βE) T(S)T [A(S)A]² 11 455, 457, 459 Q(C)R(V)T[A(C)A(V)A]² (βF) (SEQ ID NO: 22) (SEQ ID NO: 23) 12 471, 473 (βG) S(T)T[A(T)A]² 13 329-331, QKH(WL)SDR [AAA(WL)AAA]² 334-336 (SEQ ID NO: 24)(SEQ ID NO: 25) ¹loop = 1 B-strand = β ²Sequences in brackets are frommutants in which alanine residues rather than IgG sequences were used toreplace the IgE target sequence. Residues in parentheses were notaltered in these mutants.

The mutant IgEs were transiently expressed in human embryonic kidney 293cells (Gorman et al., supra), purified on a mouse anti-human IgEantibody affinity column and samples run using SDS-PAGE to ascertainthat the mutant proteins were of the proper molecular weight.

EXAMPLE 3 Soluble FCEH Binding Assay

This assay is a sequential inhibition ELISA which measures binding tothe FCEH only. In this assay, a monoclonal antibody against the FCEH iscoated onto ELISA plates at a concentration of 1 μg/ml in 50 mM sodiumcarbonate pH 9.6 for two hours at room temperature, and blocked for twohours with PBS containing 0.5% bovine serum albumin (PBSA), then washedthree times with ELISA wash buffer (0.05% Tween 20 in PBS).Recombinantly produced soluble FCEH is added at a concentration of 50units/ml and incubated for one hour, then washed five times in ELISAwash buffer. Mutant IgE samples are then added to the wells andincubated for one to two hours. The excess mutant IgE is removed byaspiration, and biotinylated IgE is then added at 50 ng/ml for 15minutes followed by five washes with ELISA wash buffer. Streptavidinconjugated to horseradish peroxidase (Sigma Chemical Company #S5512) wasadded at a 1:5000 dilution for 15 minutes, then washed three times withELISA wash buffer. Color was developed with a tetramethyl benzidineperoxidase substrate system (Kirkegaard &. Perry Labs # 50-76-00, Lot.no. NA 18) for seven minutes at 25° C. The reaction was stopped by theaddition of 1 M HCl. The ability of the mutant IgE to bind the FCEH isassessed by the degree to which the biotinylated IgE is prevented frombinding. This assay is designed to test for any FCEH binding by themutant IgE and is not meant to determine the affinity of the mutant forthe FCEH relative to native IgE.

FACS Based Binding Assays for U266 IgE Mutants

Tissue culture supernatants from 293s cells transfected with U266 IgEcDNA were harvested at either 48 or 96 hours post transfection. Tissueculture supernatants were concentrated 5-X with Amicon Centriprep 30®centrifugal concentrators (30,000 MW cutoff). Concentrated supernatantswere passed through a mouse monoclonal anti-U266 IgE affinity column(Genentech MAE1 coupled to CnBr-Sepharose). U266 IgE was eluted from thecolumn with 3.O M potassium cyanate in 50 mM tris buffer Ph 7.8. Eluatefractions containing protein as determined by O.D. 280 nm were pooledand placed in Amicon Centricon 30® concentrators. Eluate buffer wasexchanged for PBS by passing multiple volumes of PBS through theconcentrator. The final volume of affinity purified supernatant rangedfrom 0.5-1 ml. Structural integrity of recombinant IgE mutants wasanalyzed on 1-12% SDS PAGE gels and compared with U266 IgE standardobtained from the U266 cell line. Mutants were also analyzed for theability to bind to a series of monoclonal and IgE antibodies to furtherascertain proper folding and structural identity with native IgE. Theconcentration of immunoreactive IgE for each IgE mutant was determinedby a human IgE capture ELISA as follows. Nunc Immunoplate Maxisorp®plates (Nunc # 4-39451) were coated overnight at 4° C. with a Genentechmurine IgG1 anti-U266 IgE (MAE1) at 1 μg/ml in coat buffer (50 mM sodiumcarbonate buffer pH 9.6). Coat antibody was removed by three washes withELISA wash buffer (0.05% Tween 20 (US Biochemical Corporation # 20605)in PBS). Non-specific sites were blocked with ELISA diluent buffer (50mM tris buffered saline containing 0.5% BSA (Sigma Chemical Company #A-7888), 0.05% Tween 20 and 2 mM EDTA) for two hours at 25° C. on anorbital shaker. Diluent buffer was removed with 3 washes of ELISA washbuffer. Serial two-fold dilutions of IgE mutants in ELISA diluent bufferwere added to the plate. U266 IgE standard (lot 1306846) was added at1000, 500, 250, 125, 62.5, 31.3, and 15.6 ng/ml in duplicate asstandards. Samples and standard were incubated two hours at 25° C.followed by three washes with ELISA wash buffer. IgE was detected withHRP conjugated Sheep anti-human IgE (ICN # N060-050-1) at 1:8000 inELISA diluent buffer for 90 min. at 25C followed by 3 washes with ELISAwash buffer. HRP conjugate was developed with a tetramethyl benzidineperoxidase substrate system (Kirkegaard & Perry Labs. # 50-76-00, Lot.no. NA 18) for 7 minutes at 25° C. The reaction was stopped by theaddition of 1 M HCl. The reaction product was analyzed with a dualwavelength spectrophotometer at 450 nm minus absorption at 570 nm. TheU266 IgE standards were used to generate a standard curve and IgEconcentrations of the sample were extrapolated by non-parametric linearregression analysis.

FcERI alpha (+) CHO 3D10 (FCEH expressing) and FcERII(CD23) (+) IM9(FCEL expressing) B cell lines were used for the binding assays. Thestably transfected CHO (duk −) cell clone 3D10 (JBC 265, 22079-22081,1990) was maintained in Iscove's modified Dulbecco's media supplementedwith 10% heat inactivated fetal calf serum, 80 Itg/ml gentamicin sulfateand 5×10⁻⁷ M methotrexate. The IM9 human B cell myeloma ATCC® CCL 159.(Ann. N.Y. Acad. Sci. 190:221-234, 1972) was maintained in GIF basemedium with 10% heat inactivated fetal bovine serum, penicillin,streptomycin (100 units/ml) and L-glutamine (2 mM). As a positivecontrol to determine the level of CD23 on the surface of IM9 cells ineach experiment, an aliquot of cells was stained with Becton Dickinsonmurine monoclonal Leu 20 (anti-CD23) at 10 μg/ml for 30 minutes at 4° C.followed by two washes in FACS buffer. The cells were then incubatedwith FITC conjugated F(ab′)₂ affinity purified goat anti-murine IgG at 5μg/ml. Adherent CHO3D10 cells were removed from tissue culture dishes byincubation with 10 mM EDTA in PBS for 2 minutes at 37° C. Cells werecounted, then resuspended in FACS buffer (0.1% BSA, 10 mM Na azide inPBS pH 7.4) at a concentration of 5×10⁶/m]. CHO3D10 and Im9 cells(5×10⁵/aliquot) were incubated in 100 μl of FACS buffer containing U266IgE standard or IgE mutants at 2 μg/ml for 30 minutes at 4° C. in 96well microtiter plates followed by two washes with FACS buffer. As acontrol, cells were incubated in buffer alone or buffer containing 2μg/ml human IgG1 (Behring Diagnostics # 400112, lot no. 801024). Cellswere then incubated in 100 μl FACS buffer containing FITC conjugatedrabbit anti-human IgE at 20 μg/ml (Accurate Chem. Co. # AXL 475F, lot.no. 040A) for 30 minutes at 4° C. followed by 3 washes with FACS buffer.400 μl of buffer containing propidium iodide at 2 μg/ml was added to thecell suspension to stain dead cells. Cells were analyzed on a BectonDickinson FACSCAN flow cytometer. Forward light scatter and 90 degreeside scatter gates were set to analyze a homogeneous population of cellsand dead cells which stained with propidium iodide were excluded fromanalysis. FITC positive cells (IgE binding) were analyzed relative tocells stained with FITC rabbit anti-H IgE alone.

The foregoing assays were used to determine the ability of the example 2IgE analogues to bind to FCEH and FCEL. The results are set forth inTable 8.

TABLE 8 BINDING OF IGE AND IGE ANALOGUES TO FCEH AND FCEL FCEH alphaConc. % CHO FCEL (CD23) Sample/Mutant (ug/ml) 3D10 (+) % IM9 (+) U266IgE 10 90.3 92.5 U266 IgE 5 89.9 82.6 U266 IgE 0.5 59.6 4.6 U266 IgE 0.115.8 1.7  1 1.65¹ 1.7 4.3  2 1.65 34.3 48.9  3 1.65 32.3 1.2  4 1.65 4.99.2  5 1.65 60.5 73.9  6 1.65 1.4 71.6  7 1.65 76.4 4.6  8 1.65 70.316.3  9 1.65 84.2 94.3 10 1.65 67.5 84.8 11 1.65 70.8 61.5 12 1.65 84.790.3 13 1.65 85.7 96.1 dh 184 (+) 1.65 83.8 21.1 PA13² (control) 10 1.3¹Values based on quantitative Elisa. U266 was used as the standard andmurine anti-F_(Cε) monoclonal antibody to capture. ²A CDR grafted humanIgG.

Three mutant IgEs exhibited complete loss of binding to the FCEHreceptor: mutants 1, 4 and 6. Mutant 6 altered β-strand D at the end ofFcε3 close to the Fcε2 domain. Mutants 1 and 4 involved alteration oftwo Fcε3 loops which are adjacent and near the Fcε4 domain. Note thatmutant 7 is subset of mutant 1 in which the three C-terminal residues ofloop AB have been changed to alanines (Table 7, 1 vs. 7). However,mutant 7 does not affect binding to FCEH. We interpret this to mean thateither 1) FcεRI binds at least one of IgE residues 377-381 or 2) theextra residue in IgG1 loop AB (9 residues) substituted for IgE loop AB(8 residues) effected deformation of some adjacent binding determinant,possibly loop EF. That mutants 8 and 10 had no affect on FcεRI bindingmost likely means that the FCEH receptor does not protrude into thecavity bounded by loop AB and β-strand D.

Although mutant 4 had a Leu replacing Gly444 (Table 7), this should notaffect the conformation of loop EF. Residue 444 is prior to theN-terminus of this α-helix. In addition, murine IgE has a Val atposition 444 and rat IgE has an Asp. The two buried hydrophobic residuesin the middle of the α-helix, W448 and I449, are retained in thesubstituted IgG1 loop (W448, L449) as is G451 which terminated theα-helix. Hence the conformation of loop EF should be similar in IgE andIgG1.

Mutants 2 and 3 exhibited decreased binding to FCEH. Since loop BC liesnear β-strand D and loop CD is in the vicinity of loop EF, it isconceivable that one or two residues in loops BC and CD contact FCEH.

Five mutant IgEs exhibited loss of binding to the FCEL receptor: mutants1, 3 4, 7 and 8. Mutants 1 and 4 were discussed above. Mutant 3 involvedalteration of loop CD; in contrast to FCEH, loop CD evidently plays amajor role in FCEL binding. Mutant 7, a subset of mutant 1 as discussedabove, comprises the C-terminal portion of loop AB and is proximal toloop EF. Additionally, mutant 8 consists of replacement of two Thrresidues (387,389) with Ala; these two residues are part of β-strand Bwhich is at the bottom of the aforementioned cavity bounded by loop ABand β-strand D. Mutant 10 comprised two different residues in thiscavity (438,440) on β-strand E, which is adjacent to β-strand B. Sincemutant 10 did not affect FCEL binding, we conclude that the FCELreceptor should have only a minimal incursion into cavity while the highaffinity receptor does not intrude into the cavity.

In addition to a glycosylation site at Asn430 which corresponds to theglycosylation site in IgG Fc, human IgE contains another glycosylationsite at Asn403. Mutant 9 converted Asn403 and Thr405 to alanines (Table6). Loss of carbohydrate did not affect binding to either receptor.

Based on the information from mutants 1-13, we propose that FCEH andFCEL have binding sites on IgE Fc which are distinct but overlap. Thelow affinity receptor seems to interact with a relatively smallerportion of the IgE Fcε3 domain involving three adjacent loops: AB, CDand EF. In contrast, the high affinity receptor interacts with a largerportion of IgE Fcε3, which spans loop EF, β-strand D and, possibly, theN-terminal portion of loop AB. Portions of loops BC and CD in thevicinity of loop EF and β-strand D may also interact with FCEH. Inaddition, FCEL may protrude into the cavity bounded by loop AB andβ-strand D whereas FCEH does not do so. Since we have not evaluated anymutants in FCε4 and only one in Fcε2 (mutant 13), it is possible thatportions of these two domains play a role in IgE-receptor binding.

EXAMPLE 4 Preparation of Humanized MaE11

Residues were selected from MaE11 and inserted or substituted into ahuman Fab antibody background (V_(H) region Kabat subgroup III and V_(L)region kappa subgroup I). A first version, humae11v1 or version 1, isdescribed in Table 9.

TABLE 9 Changes in V_(H) human subgroup III and V_(L) κ subgroup I(Kabat) consensus sequences for humanized MaE11 Version 1 Hu Residue CDRCDR Domain Residue No. V.1 by Kabat by Chothia V_(L) M  4 L Insert30abcd YDGD L1 L1 (SEQ ID NO: 26) L* 33 M L1 S 53 Y L2 Y 91 S L3 L3 N 92H L3 L3 S 93 E L3 L3 L 94 D L3 L3 V_(H) A 24 V F* 27 Y H1 H1 T 28 S H1H1 F* 29 I H1 H1 Insert 29a T H1 H1 D 31 G H1 H1 A 33 S H1 H1 M* 34 W H1H1 V 37 I V 50 S H2 S 52 T H2 N 53 Y H2 H2 G 54 D H2 H2 S 55 G H2 H2 Y58 N H2 L 78 F D 95 G H3 97-101 All Changed H3 H3 to MaE11 Sequence*These residues typically do not vary despite their position withinCDRs. The remaining residues found in the KI and III CDR sequences(particularly the CDRs by Chothia structural analysis), will vary widelyamong recipient human antibodies.

The affinity of version 1 was assayed and found to be about 100 timeslower than that of the donor antibody Mae11 (see FIGS. 4 a and 4 b).Therefore, further modifications in the sequence of version 1 were madeas shown in Table 10. Determination was made of the ability of thesefurther modifications to inhibit the binding of labelled huIgE to FCEH.

The 50% inhibition assays whose results are shown in Table 10 wereconducted as follows:

A 96-well assay plate (Manufn Nunc.) was coated with 0.05 ml of theFcεRI alpha chain IgG1 chimeric receptor in 1 μg/ml coating buffer (50nmol carbonate/bicarbonate, pH 9.6). Assay was done for 12 hours at 4-8°C. The wells were aspirated and 250 μl blocking buffer (PBS—1% BSA pH7.2) was added and incubated for one hour at 4° C. In a separate assayplate the samples and reference murine MaE11 antibody were titered from200 μg/ml by 1 to 10-fold dilution with assay buffer (0.5% BSA, 0.05%Tween 20, PBS, pH 7.2) and an equal volume of 10 ng/ml biotinylated IgEat 10 ng/ml was added and the plate incubated for 2-3 hours at 25° C.The FcεRI-coated wells were washed three times with PBS-0.05% Tween20,and then 50 μl from the sample wells were transferred and incubated withagitation for 30 minutes at 25° C. 50 μl/well of streptavidin-HRPdiluted 1:5000 in assay buffer was incubated for 15 minutes withagitation and then the plate was washed as before. 50 μl/well ofMicrowell peroxidase substrate (Kirkgaard & Parry Laboratories) wasadded and color was developed for 30 minutes. The reaction was stoppedby adding an equal volume of 1 normal HCl and the adsorbance measured at450 nm. The concentration for 50% inhibition was calculated by plottingpercent inhibition versus concentration of blocking antibody with anonlinear 4-parameter curve-fit for each antibody using INPLOT.

TABLE 10 Humanized MaE11 Variants Conc. At 50% S.D. for Version Changesfrom inh. (ng/ml)* prev. F(ab)-X/ [F(ab)-X] Domain F(ab)-Version 1Purpose Mean col. F(ab)-1  1 — — — 6083 1279 1.0  2 V_(L) L4M Packing;CDR-L1 9439 508 1.6 M33L  3 V_(L) E55G Sequence usually 5799 523 1.0G57E E55-X-G57  4 V_(H) I37V VL-VH interface 8622 107 1.4  5 V_(H) V24APacking; CDR-H1 9387 733 1.6  6 V_(H) F78L Packing; CDR- 17537 4372 2.9H1, H2  7 V_(L) L4M remake version 1 to >100000 >16.0# V_(H) R24Kaccomplish a E55G direct exchange of G57E CDR residues V24A I37V T57SA60N D61P V63L G65N F78L  7a V_(H) As V.7 except V_(H) 98000 16.0 L78 isF  8 V_(H) A60N Extended Kabat 1224 102 0.20 D61P CDR-H2 & A60N is atV_(L)-V_(H) interface  8a V_(H) As V.8 except V_(H) CDR-H2; packing 41666 0.07 V63 is L and F67 of L63 and I67 is I  8b V_(H) As V.8 except F67CDR-H2; packing 501 84 0.08 is I of V63 and I67  9 V_(L) A13V RepackVersion 1 842 130 0.14 V_(H) V19A interior as in V58I murine MaE11 L78VV104L V48M A49G A60N V63L F67I I69V M82L L82cA 23 V_(L) L4M Packing;CDR-L1 6770 349 1.1  1 — — — 6083 1279 1.0 10 V_(L) D30ACDR-L1 >100000 >16.0 D30bA modification D30dA 11 V_(L) E93A CDR-L3 174567115 2.9 D94A modification 12 V_(H) D54A CDR-H2 2066 174 0.34modification 13 V_(H) H97A CDR-H3 >100000 >16.0 H100aA modificationH100cA 14 V_(L) D30A CDR-L1 3452 183 0.57 modification 15 V_(L) D30bACDR-L1 6384 367 1.0 modification 16 V_(L) D30dA CDR-L1 >100000 >16.0modification 17 V_(H) H97A CDR-H3 19427 8360 3.2 modification 18 V_(H)H100aA CDR-H3 2713 174 0.45 modification 19 V_(H) H100cA CDR-H3 158468128 2.6 modification *Inhibition of fitc-IgE binding to FCEH (FcERI).Full-length antibody and humanized fragment versions: mean and standarddeviation of three assays. #A F(ab)-X/F(ab)-1 ratio of >16 means thatthis variant exhibited no binding even at the highest F(ab)concentrations used.

As can be seen from Table 10 and FIGS. 4 a and 4 b, version 8 (in whichhuman residues of version 1 at sites 60 and 61 in the light chain werereplaced by their Mae11 counterparts) demonstrated substantiallyincreased affinity. Further increases in affinity are seen in versions8a and 8b, where one or two murine residues replaced human residues.Other increases, at least virtually to the level of Mae11, wereaccomplished by replacing hydrophobic human residues found in theinterior of V_(H) and V_(H1) with their MaE11 counterparts, resulting inthe variant designated version 9 (see Table 10 and FIGS. 4 a and 4 b).Accordingly, the humanized antibodies of this invention will possessaffinities ranging about from 0.1 to 100 times that of MAE11.

Table 11 explores the effects on FCEH affinity of various combinationsof humanized maE11 IgG1 variants.

TABLE 11 Humanized MaE11 IgG1 Variants Conc. at 50% S.D. from inh.(ng/ml) previous Var. X/ Var. X/ Variant Mean* column* IgL1H1 MaE11IgL1H1 7569 1042 1.0 16.9 IgL1H8 3493 1264 0.46 7.8 IgL9H9 1118 172 0.152.5 IgL1H9 608 364 0.08 1.4 IgL9H1 5273 2326 0.70 11.7 IgL1H8b 1449 2260.19 3.2 MaE11 449 53 0.06 1.0 *L1 = V_(L) as in F(ab)-1 (human buriedresidues--not exposed to solvent); L9 = V_(L) as in F(ab)-9 (murineburied residues); H1 = V_(H) as in F(ab)-1 (human buried residues); H8 =V_(H) as in F(ab)-8 (F(ab)-1 with AlaH60Asn, AspH61Pro); H9 = V_(H) asin F(ab)-9 (murine buried residues); H8b = V_(H) as in F(ab)-8b (F(ab)-8with PheH67Ile).

EXAMPLE 5 Creation of IgE Mutants

IgE mutants (Table 12) were prepared to evalute their effect on bindingto anti-IgE, especially MaE11, and to FcεRI and FcεRII. Some of themutants were designed to substitute for a specific amino acid residueanother residue with either similar or very different charge or size.The impact of these changes on receptor binding is reflected in thetable below.

The receptor assays are performed substantially as follows:

A 96-well assay plate (Manufn Nunc.) was coated with 0.05 ml of FcεRI orRII IgG1 chimeric receptor in 1 μg/ml coating buffer (50 nmolcarbonate/bicarbonate, pH 9.6). Assay was done for 12 hours at 4-8° C.The wells were aspirated and 250 μl blocking buffer (PBS—1% BSA pH 7.2)was added and incubated for one hour at 4° C. In a separate assay platethe samples and reference murine MaE11 antibody were titered from 200μg/ml by 1 to 10-fold dilution with assay buffer (0.5% BSA, 0.05% Tween20, PBS, pH 7.2) and an equal volume of 10 ng/ml biotinylated IgE at 10ng/ml was added and the plate incubated for 2-3 hours at 25° C. TheFcεRI-coated wells were washed three times with PBS-0.05% Tween 20, andthen 50 μl from the sample wells were transferred and incubated withagitation for 30 minutes at 25° C. 50 μl/well of streptavidin-HRPdiluted 1:5000 in assay buffer was incubated for 15 minutes withagitation and then the plate was washed as before. 50 μl/well ofMicrowell peroxidase substrate (Kirkgaard & Parry Laboratories) wasadded and color was developed for 30 minutes. The reaction was stoppedby adding an equal volume of 1 normal HCl and the adsorbance measured at450 nm. The absorbance was plotted versus concentration of blockingantibody MaE11 and an inhibition standard curve was generated usingINPLOT.

TABLE 12 Amino acid sequences of IgE mutants Kabat Human IgE Fce3 MutantResidue # seq. Mutant seq. Fcε-RI* FcεRII* Loop AB 1 377-385 FDLFIRKSKDTLMISRT − − (SEQ ID NO: 27) (SEQ ID NO: 28) 7 383-385 RKS AAA +/−, −+, − 21 377, 381 F(DL)F Q(DL)H + + (SEQ ID NO: 29) (SEQ ID NO: 30) 66382 I A + + 67 383 R A + +/− 68 384 K A + + 69 385 S A 102 383, 384 RKDD β-strand B 8 387, 389 T(I)T A(I)A +/−, + − 70 387 T A + +/−, + 71 389T A + + Loop BC 2 396-401 APSKGT SHEDPQ (SEQ ID NO: 31) (SEQ ID NO: 32)β-strand C 9 403, 405 N(L)T A(L)A + + Loop CD 3 407-420 SRASGKPVNHSYVDGVQVHNAK +/− − (SEQ ID NO: 33) (SEQ ID NO: 34) 55 407-415 SR(A)S(G)KAA(A)A(G)A +/− + (SEQ ID NO: 35) (SEQ ID NO: 36) 59 407 S A + + 60 408 RA + − 61 411 S A + + 62 415 K A + − 63 418 N A +/− + 64 419 H A + + 65420 S A +/− + 100 408 R E 101 415 K D β-strand D 6 423-428 KEEKQRPREQQY + + (SEQ ID NO: 37) (SEQ ID NO: 38) 35 422 R A + + 36 4423 KA + + 37 424 E A + + 38 425 E A + + 39 426 K A + 40 427 Q A −, +/− + 41428 R A + + 75 423-425 KEE AAA −, +/−, + + 76 426-428 KQR AAA 79 423,425, 427 KEEKQR AEAKAR (SEQ ID NO: 39) (SEQ ID NO: 40) 80 424, 426, 428KEEKQR KAEAQA (SEQ ID NO: 41) (SEQ ID NO: 42) 81 423, K P 82 423-427KEEKQR AAEAQA (SEQ ID NO: 43) (SEQ ID NO: 44) β-strand E 10 438, 440T(S)T A(S)A + + Loop EF 4 444-453 GTRDWIEGET LHQDWLDGKE − − (SEQ ID NO:45) (SEQ ID NO: 46) 49 445 T A + + 50 336 R A + − 51 337 D A + +, − +/−52 450 E A + − 53 452 E A + + + +/− 77 445, 446 TR AA − − 78 450, 452,453 E(G)ET A(G)AA + + (SEQ ID NO: 47) (SEQ ID NO: 48) + 83 444 G L + +84 444 G A 85 445-453 TRDWIEGET HQDWLDGKE − + (SEQ ID NO: 49) (SEQ IDNO: 50) + 86 445 T H + 87 445, 446 TR HQ +/−, + 88 446 R E − 89 450,452, 453 E(G)ET D(G)KE +/−, − +/− (SEQ ID NO: 51) (SEQ ID NO: 52) + 93447 D R +/−, − 94 450 E R 95 452 E R 96 453 T R 97 447 D N 98 452 E Q 99452 E D β-strand F 11 445, 457, 459 Q(C)R(V)T A(C)A(V)A (SEQ ID NO: 53)(SEQ ID NO: 54) Loop FG 5 465-469 RALM APIE (SEQ ID NO: 55) (SEQ ID NO:56) β-strand G 12 471, 473 S(T)T A(T)A +, + Fcε2 13 329-331, 334-QKH(WL)SDR AAA(WL)AAA +, + 336 (SEQ ID NO: 57) (SEQ ID NO: 58) Fcε4 72498-501 PRAA QPRE (SEQ ID NO: 59) (SEQ ID NO: 60) 73 594-599 ASPSQTLHNHY (SEQ ID NO: 61) (SEQ ID NO: 62) 74 595-599 S(P)SQT A(P)AAA (SEQ IDNO: 63) (SEQ ID NO: 64) *Positive receptor binding indicated by “+”, nobinding by “−”, and positive binding but less than unaltered is shown by“+/−”. Where more than one assay was performed, results are separated bycommas.

1. An isolated nucleic acid molecule encoding a humanized antibody thatspecifically binds IgE comprising: (a) a VH domain and a VL domain,wherein: (i) the VH domain comprises the VH domain of SEQ ID NO:8 inwhich the alanine residue at position 61 of SEQ ID NO:8 is replaced byan asparagine residue, the aspartic acid residue at position 62 of SEQID NO:8 is replaced by a proline residue, the valine residue at position64 of SEQ ID NO:8 is replaced by a leucine residue, and thephenylalanine residue at position 68 of SEQ ID NO:8 is replaced by anisoleucine residue, in which said positions correspond to Kabatnumbering 60, 61, 63 and 67, respectively, and (ii) the VL domaincomprises the VL domain of SEQ ID NO:9; or (b) a VH domain and a VLdomain, wherein: (i) the VH domain comprises the VH domain of SEQ IDNO:8 in which the alanine residue at position 61 of SEQ ID NO:8 isreplaced by an asparagine residue, the aspartic acid residue at position62 of SEQ ID NO:8 is replaced by a proline residue, and thephenylalanine residue at position 68 of SEQ ID NO:8 is replaced by anisoleucine residue, in which said positions correspond to Kabatnumbering 60, 61 and 67, respectively, and (ii) the VL domain comprisesthe VL domain of SEQ ID NO:9; or (c) a VH domain and a VL domain,wherein: (i) the VH domain comprises the VH domain of SEQ ID NO:8 inwhich the valine residue at position 49 of SEQ ID NO:8 is replaced by amethionine residue, the alanine residue at position 50 of SEQ ID NO:8 isreplaced by a isoleucine residue, the alanine residue at position 61 ofSEQ ID NO:8 is replaced by an asparagine residue, the valine residue atposition 64 of SEQ ID NO:8 is replaced by an leucine residue, thephenylalanine residue at position 68 of SEQ ID NO:8 is replaced by anisoleucine residue, the isoleucine residue at position 70 of SEQ ID NO:8is replaced by a valine residue, the methionine residue at position 83of SEQ ID NO:8 is replaced by a leucine residue, and the leucine residueat position 86 of SEQ ID NO:8 is replaced by an alanine residue, inwhich said positions correspond to Kabat numbering 48, 49, 60, 63, 67,69, 82 and 82c, respectively, and (ii) the VL domain comprises the VLdomain of SEQ ID NO:9 in which the alanine residue at position 13 of SEQID NO:9 is replaced by a valine residue, the valine residue at position19 of SEQ ID NO:9 is replaced by an alanine residue, the valine residueat position 62 of SEQ ID NO:9 is replaced by an isoleucine residue, theleucine residue at position 82 of SEQ ID NO:9 is replaced by a valineresidue, and the valine residue at position 108 of SEQ ID NO:9 isreplaced by a leucine residue, in which said positions correspond toKabat numbering 13, 19, 58, 78 and 104, respectively.
 2. The nucleicacid of claim 1, wherein the encoded antibody is an IgG1 antibody. 3.The nucleic acid of claim 1, wherein the encoded antibody is an IgG2antibody.
 4. The nucleic acid of claim 1, wherein the encoded antibodyis an IgG3 antibody.
 5. The nucleic acid of claim 1, wherein the encodedantibody is an IgG4 antibody.
 6. A vector comprising the nucleic acid ofclaim
 1. 7. A mammalian host cell comprising the vector of claim
 6. 8. Amethod of producing the humanized antibody of claim 1 comprisingculturing a mammalian host cell that comprises a vector comprisinp thenucleic acid of claim 1 and recovering said antibody.
 9. An isolatednucleic acid molecule encoding a humanized antibody that specificallybinds to IgE comprising a VH domain and a VL domain, wherein: (a) the VHdomain comprises the VH domain of SEQ ID NO:8 in which the alanineresidue at position 61 of SEQ ID NO:8 is replaced by an asparagineresidue, the aspartic acid residue at position 62 of SEQ ID NO:8 isreplaced by a proline residue, the valine residue at position 64 of SEQID NO:8 is replaced by a leucine residue, and the phenylalanine residueat position 68 of SEQ ID NO:8 is replaced by an isoleucine residue, inwhich said positions correspond to Kabat numbering 60, 61, 63 and 67,respectively and (b) the VL domain comprises the VL domain of SEQ IDNO:9.
 10. An isolated nucleic acid molecule encoding a polypeptidecomprising a VH domain and a VL domain, wherein: (a) the VH domaincomprises the VH domain of SEQ ID NO:8 in which the alanine residue atposition 61 of SEQ ID NO:8 is replaced by an asparagine residue, theaspartic acid residue at position 62 of SEQ ID NO:8 is replaced by aproline residue, the valine residue at position 64 of SEQ ID NO:8 isreplaced by a leucine residue, and the phenylalanine residue at position68 of SEQ ID NO:8 is replaced by an isoleucine residue, in which saidpositions correspond to Kabat numbering 60, 61, 63 and 67, respectivelyand (b) the VL domain comprises the VL domain of SEQ ID NO:9.
 11. Thenucleic acid of claim 9, wherein the encoded antibody is an IgG1antibody.
 12. The nucleic acid of claim 9, wherein the encoded antibodyis an IgG2 antibody.
 13. The nucleic acid of claim 9, wherein theencoded antibody is an IgG3 antibody.
 14. The nucleic acid of claim 9,wherein the encoded antibody is an IgG4 antibody.
 15. The nucleic acidof claim 10, wherein the encoded polypeptide further comprises an IgG1constant domain.
 16. The nucleic acid of claim 10, wherein the encodedpolypeptide further comprises an IgG2 constant domain.
 17. The nucleicacid of claim 10, wherein the encoded polypeptide further comprises anIgG3 constant domain.
 18. The nucleic acid of claim 10, wherein theencoded polypeptide further comprises an IgG4 constant domain.
 19. Avector comprising the nucleic acid of claim
 9. 20. A vector comprisingthe nucleic acid of claim
 10. 21. A mammalian host cell comprising thevector of claim
 19. 22. A method of producing the humanized antibody ofclaim 9 comprising culturing a mammalian host cell that comprises avector comprising the nucleic acid of claim 9 and recovering saidantibody.
 23. A mammalian host cell comprising the vector of claim 20.24. An isolated nucleic acid molecule encoding a humanized antibody thatspecifically binds to IgE comprising a VH domain and a VL domain,wherein: (a) the VH domain comprises the VH domain of SEQ ID NO:8 inwhich the alanine residue at position 61 of SEQ ID NO:8 is replaced byan asparagine residue, the aspartic acid residue at position 62 of SEQID NO:8 is replaced by a proline residue, and the phenylalanine residueat position 68 of SEQ ID NO:8 is replaced by an isoleucine residue, inwhich said positions correspond to Kabat numbering 60, 61 and 67,respectively, and (b) the VL domain comprising the VL domain of SEQ IDNO:9.
 25. An isolated nucleic acid molecule encoding a polypeptidecomprising a VH domain and a VL domain, wherein: (a) the VH domaincomprises the VH domain of SEQ ID NO:8 in which the alanine residue atposition 61 of SEQ ID NO:8 is replaced by an asparagine residue, theaspartic acid residue at position 62 of SEQ ID NO:8 is replaced by aproline residue, and the phenylalanine residue at position 68 of SEQ IDNO:8 is replaced by an isoleucine residue, in which said positionscorrespond to Kabat numbering 60, 61 and 67, respectively, and (b) theVL domain comprising the VL domain of SEQ ID NO:9.
 26. The nucleic acidof claim 24, wherein the encoded antibody is an IgG1 antibody.
 27. Thenucleic acid of claim 24, wherein the encoded antibody is an IgG2antibody.
 28. The nucleic acid of claim 24, wherein the encoded antibodyis an IgG3 antibody.
 29. The nucleic acid of claim 24, wherein theencoded antibody is an IgG4 antibody.
 30. The nucleic acid of claim 25,wherein the encoded polypeptide further comprises an IgG1 constantdomain.
 31. The nucleic acid of claim 25, wherein the encodedpolypeptide sequenoe further comprises an IgG2 constant domain.
 32. Thenucleic acid of claim 25, wherein the encoded polypeptide furthercomprises an IgG3 constant domain.
 33. The nucleic acid of claim 25,wherein the encoded polypeptide further comprises an IgG4 constantdomain.
 34. A vector comprising the nucleic acid of claim
 24. 35. Avector comprising the nucleic acid of claim
 25. 36. A mammalian hostcell comprising the vector of claim
 34. 37. A method of producing thehumanized antibody of claim 24 comprising culturing a mammalian hostcell that comprises a vector comprising the nucleic acid of claim 24 andrecovering said antibody.
 38. A mammalian host cell comprising thevector of claim
 35. 39. An isolated nucleic acid molecule encoding ahumanized antibody that specifically binds to IgE comprising a VH domainand a VL domain, wherein: (a) the VH domain comprises the VH domain ofSEQ ID NO:8 in which the valine residue at position 49 of SEQ ID NO:8 isreplaced by a methionine residue, the alanine residue at position 50 ofSEQ ID NO:8 is replaced by a glycine residue, the alanine residue atposition 61 of SEQ ID NO:8 is replaced by an asparagine residue, thevaline residue at position 64 of SEQ ID NO:8 is replaced by a leucineresidue, the phenylalanine residue at position 68 of SEQ ID NO:8 isreplaced by an isoleucine residue, the isoleucine residue at position 70of SEQ ID NO:8 is replaced by a valine residue, the methionine residueat position 83 of SEQ ID NO:8 is replaced by a leucine residue, and theleucine residue, at position 86 of SEQ ID NO:8 is replaced by an alanineresidue, in which said positions correspond to Kabat numbering 48, 49,60, 63, 67, 69, 82 and 82c, respectively, and (b) the VL domaincomprises the VL domain of SEQ ID NO:9 in which the alanine residue atposition 13 of SEQ ID NO:9 is replaced by a valine residue, the valineresidue at position 19 of SEQ ID NO:9 is replaced by an alanine residue,the valine residue at position 62 of SEQ ID NO:9 is replaced by anisoleucine residue, the leucine residue at position 82 of SEQ ID NO:9 isreplaced by a valine residue, and the valine residue at position 108 ofSEQ ID NO:9 is replaced by a leucine residue, in which said positionscorrespond to Kabat numbering 10, 19, 58, 78 and 104, respectively. 40.An isolated nucleic acid molecule encoding a polypeptide comprising a VHdomain and a VL domain, wherein: (a) the VH domain comprises the VHdomain of SEQ ID NO:8 in which the valine residue at position 49 of SEQID NO:8 is replaced by a methionine residue, the alanine residue atposition 50 of SEQ ID NO:8 is replaced by a glycine residue, the alanineresidue at position 61 of SEQ ID NO:8 is replaced by an asparagineresidue, the valine residue at position 64 of SEQ ID NO:8 is replaced bya leucine residue, the phenylalanine residue at position 68 of SEQ IDNO:8 is replaced by an isoleucine residue, the isoleucine residue atposition 70 of SEQ ID NO:8 is replaced by a valine residue, themethionine residue at position 83 of SEQ ID NO:8 is replaced by aleucine residue, and the leucine residue at position 86 of SEQ ID NO:8is replaced by an alanine residue, in which said positions correspond toKabat numbering 48, 49, 60, 63, 67, 69, 82 and 82c, respectively, and(b) the VL domain comprises the VL domain of SEQ ID NO:9 in which thealanine residue at position 13 of SEQ ID NO:9 is replaced by a valineresidue, the valine residue at position 19 of SEQ ID NO:9 is replaced byan alanine residue, the valine residue at position 62 of SEQ ID NO:9 isreplaced by an isoleucine residue, the leucine residue at position 82 ofSEQ ID NO:9 is replaced by a valine residue, and the valine residue atposition 108 of SEQ ID NQ:9 is replaced by a leucine residue, in whichsaid positions correspond to Kabat numbering 13, 19, 58, 78 and 104,respectively.
 41. The nucleic acid of claim 39, wherein the encodedantibody is an IgG1 antibody.
 42. The nucleic acid of claim 39, whereinthe encoded antibody is an IgG2 antibody.
 43. The nucleic acid of claim39, wherein the encoded antibody is an IgG3 antibody.
 44. The nucleicacid of claim 39, wherein the encoded antibody is an IgG4 antibody. 45.The nucleic acid of claim 40, wherein the polypeptide further comprisesan IgG1 constant domain.
 46. The nucleic acid of claim 40, wherein theencoded polypeptide further comprises an IgG2 constant domain.
 47. Thenucleic acid of claim 40, wherein the encoded polypeptide furthercomprises an IgG3 constant domain.
 48. The nucleic acid of claim 40,wherein the encoded polypeptide further comprises an IgG4 constantdomain.
 49. A vector comprising the nucleic acid of claim
 39. 50. Avector comprising the nucleic acid of claim
 40. 51. A mammalian hostcell comprising the vector of claim
 49. 52. A method of producing thehumanized antibody of claim 39 comprising culturing a mammalian hostcell that comprises a vector comprising the nucleic acid of claim 39 andrecovering said antibody.
 53. A mammalian host cell comprising thevector of claim 50.